INHIBITION OF THE ACTIVITY OF KINASE AND SYNTHETASE ENZYMES

Abstract

The invention provides a method of inhibiting the activity of a kinase or a synthetase, the method including binding an active site of the kinase or synthetase with a deuterated imidazole moiety, thereby inhibiting the activity of the kinase or the synthetase.

Description

[0001] THIS INVENTION relates to the inhibition of the activity of kinase and synthetase enzymes. More particularly, the invention relates to a method of inhibiting the activity of a kinase or a synthetase, to a method of coupling a kinase or a synthetase to a nucleotide, to a method of generating a compound that inhibits the activity of a kinase or a synthetase, to a computer-assisted method of generating a test inhibitor of the activity of a kinase or a synthetase, and to a method of screening a test compound in vitro to determine whether or not it inhibits the activity of a kinase or a synthetase.

[0002] Adenylate kinase (AK) contributes to the homeostasis of adenine nucleotides by maintaining intracellular nucleotide pools. Six isoenzymes of adenylate kinase have been identified in mammalian cells with different subcellular localization and substrate specificity. Adenylate kinase catalyses the reaction:

ATP+AMP→2ADP

where ATP is adenosine triphosphate, AMT is adenosine monophosphate and ADP is adenosine diphosphate. The adenylate kinases (ATP:AMP phosphotransferases, EC 2.7.4.3) catalyze the reversible transfer of the γ-phosphate group from a phosphate donor (ATP, GTP, CTP, ITP) to AMP.

[0003] The phosphate donor is usually ATP. There is a size variation among the isoenzymes: AK1, AK5 and AK6 are short type adenylate kinases while AK2, AK3 and AK4 are long type adenylate kinases containing a 27 amino acid insertion sequence in the central portion of the peptide. The mammalian adenylate kinases have a distinct intracellular compartmentalization, with AK1 in the cytosol, AK2 in the inter-membrane space of mitochondria, AK3 in the mitochondrial matrix, AK4 being mitochondrial in nature, AK5 (unknown localization) and AK6 in the nucleus. AK3 present in the mitochondrial matrix has a preference for GTP over ATP.

[0004] It is often desired to regulate the activity of kinases and synthetases, and this can, for example, be done by binding an active site of a kinase, such as AK, or a synthetase, such as glutamine synthetase, to an imidazole moiety, eg as found in ATP. An object of this invention is to provide a means whereby the level of inhibition of the activity of a kinase or a synthetase can be enhanced.

[0005] Thus, according to a first aspect of the invention, there is provided a method of inhibiting the activity of a kinase or a synthetase, the method including binding an active site of the kinase or synthetase with a deuterated imidazole moiety, thereby inhibiting the activity of the kinase or the synthetase.

[0006] The method may be applied to a kinase. Examples of suitable kinases are adenylate kinase (AK), shikimate kinase (SK), pyruvate kinase (PK), hexokinase (HXK), aspartokinase (ASK), creatine kinase (CK), glycerate kinase, acetate kinase, glutamine synthetase, such as adenylated glutamineate synthetase, and/or deadenylated glutamine synthetase, phosphofructokinase, and isoforms thereof. In a particular embodiment of the invention, the kinase may be adenylate kinase (AK). Its sequence comprises conserved residues Arg 97, Glu 98, Arg 128 and Asp 180.

[0007] Instead, the method may be applied to a synthetase. The synthetase may then, for example, be glutamine synthetase (GS).

[0008] Generally, the kinase or the synthetase, or an isoenzyme thereof, may comprise amino acid residues identical to, or similar to, conserved adenylate kinase residues Arg 97, Glu 98, Arg 128 and Asp 180, at positions equivalent to the positions of these residues in adenylate kinase.

[0009] The inhibition of the kinase or the synthetase activity may be effected in vitro or in vivo.

[0010] More particularly, the deuterated imidazole moiety may be provided by a nucleotide. In one embodiment of the invention, the nucleotide may be adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP), with the nucleotide having an immonium moiety induced at position N7, so that a carbene is induced at the C8 position, and with deuteration being effected at the C8 position. However, in another embodiment of the invention, the nucleotide may be a compound which is deuterated at a position equivalent to C8 in ATP, ADP and AMP.

[0011] The extent of regulation of each form (isoenzymes) of the kinase enzyme or the synthetase enzyme is different based on the level of the Kinetic Isotope Effect (KIE) and the kinetics of the enzymes and the structure of the active site of the kinase. When the kinase is adenylate kinase, the reaction catalysed by adenylate kinase is:

ATP+AMP→2ADP

[0012] The assay is carried out in the presence of ATP and AMP measuring the formation of ADP. The KIE is obtained when comparing the enzyme activity of the adenylate kinase enzyme when the reaction is carried out in the presence of ATP and deuterated ATP. A kinetic isotope effect is also obtained when comparing the activity of the enzyme when the reaction is carried out in the presence of AMP and deuterated AMP. A further KIE is obtained when both deuterated ATP and deuterated AMP are used in the reaction. It is significant that for all these enzymes, the KIE occurs as a result of the change in using ATP over deuterated ATP or AMP over deuterated AMP. The KIE is obtained by dividing the activity of the enzyme in the presence of the proteated species (v_H) by the enzyme activity in the presence of the deuterated species (v_D) and is v_H/v_D=0.5. The KIE is found over a broad range of ATP concentration enzyme activity profile. The extent of the regulation of AK is also ATP concentration dependent. The proposed basic reaction mechanisms by which all forms of kinase are regulated occurs via either the formation of an immonium species at N7, which in turn induces the formation of a carbene at C8 or via the delocalization of electrons away from the C8 rendering the C8 more acidic. An immonium species may be induced at N7 by (1) protonation via a coordinated HCO₃^-, (2) carboxylation of the N7, or (3) the coordination of an amino acid side chain within the active site such as a arginine, glutamine, lysine or histidine. The different forms of regulation are then based on the mechanism by which the carbene formed at C8 is stabilized and the mechanism by which the C8-H is deprotonated or via the interaction of the protein coordinated amino acid side chains affecting the delocalization of electrons around the adenyl ring of the nucleotide.

[0013] The formation of the immonium species at N7 then facilitates the deprotonation of C8 via a coordinated amino acid residue. The resulting carbene intermediate is stabilised by the putative bond formation by a coordinated amino acid and C8. In some kinases the coordination may be mediated by the presence of a divalent metal ion such as Mn²+ coordinated into the ATP coordination complex.

[0014] In particular, it was found that the activity of AK1 is affected by the deuteration of ATP and AMP at the C8 position. The role of deuteration of ATP/AMP in causing a KIE in AK1 demonstrates that the binding mechanism of nucleotides to the active sites of kinases is similar and the imidazole moiety is implicated in all cases. "Imidazole" moiety-containing compounds are found to affect the regulation of AK1 and when these compounds are deuterated at the position in the imidazole moiety equivalent to the C8 position in ATP.

[0015] Thus, in accordance with the invention, the nucleotides ATP, ADP and AMP will be bound to the AK active site, with the nucleotide being either protonated or unprotonated at position N7. The protonated form is positively charged while the unprotonated form is neutral. The unprotonated from may be protonated within the active site of the enzyme also inducing the formation of an immonium species. In both cases, ie when the nucleotide is in either the protonated form or the neutral form, an immonium species at the N7 position facilitates the induction of a carbene at the C8 position.

[0016] In the case of the neutral form of the nucleotide, the method may include creating an immonium species at position N7 through the donation of a protein by the AK enzyme. This facilitates the induction of the carbene at the C8 position by making the C8-H more acidic.

[0017] The rendering of the C8-H to become more acidic via the coordination of amino acid side chains and the delocalization of electrons away from C8 is another mechanism by which the regulation of kinases occurs.

[0018] According to a second aspect of the invention, there is provided a method of coupling a kinase or a synthetase to a nucleotide or to a nucleotide analogue, to inhibit the activity of the kinase or the synthetase, the method including binding an active site of the kinase or the synthetase with a nucleotide or with a nucleotide analogue comprising an imidazole moiety in deuterated form.

[0019] According to a third aspect of the invention, there is provided a method of generating a compound that inhibits the activity of a kinase or a synthetase, the method comprising providing a three-dimensional structure of a kinase or a synthetase; and designing, based on the three-dimensional structure, a compound capable of inhibiting the activity of the kinase or the synthetase, the compound comprising a deuterated imidazole moiety.

[0020] The compound may be a nucleotide or imidazole containing compound as hereinbefore described.

[0021] According to a fourth aspect of the invention, there is provided a computer-assisted method of generating a test inhibitor of the activity of a kinase or a synthetase, the method using a processor and an input device, the method comprising

[0022] (a) inputting, on the input device, data comprising a structure of a kinase or a synthetase;

[0023] (b) docking into an active site of the kinase or the synthetase, a test inhibitor molecule comprising a deuterated imidazole moiety, using the processor; and

[0024] (c) determining, based on the docking, whether the test inhibitor compound would inhibit the kinase or synthetase activity.

[0025] The method may include determining, based on the docking, whether the test inhibitor molecule would inhibit the transfer of a y-phosphate group from a phosphate donor.

[0026] The method may further comprise designing a test inhibitor determined by step (c) to inhibit the kinase or the synthetase activity and evaluating the inhibitory activity of the test inhibitor on a bacterial kinase or synthetase in vitro.

[0027] According to a fifth aspect of the invention, there is provided a method of screening a compound in vitro to determine whether or not it inhibits the activity of kinase or a synthetase, the method comprising contacting a kinase or a synthetase with a compound comprising a protonated imidazole moiety; contacting the kinase or the synthetase with the same compound comprising a deuterated imidazole moiety; and determining whether or not the activity of the kinase or synthetase is reduced in the presence of the compound containing the deuterated imidazole moiety relative to the activity of the same kinase or synthetase in the presence of the compound containing the protonated imidazole moiety.

[0028] The method of the second, third, fourth or fifth aspect may be applied to a kinase. Examples of suitable kinases are then, as hereinbefore described, adenylate kinase (AK), shikimate kinase (SK), pyruvate kinase (PK), hexokinase (HXK), aspartokinase (ASK), creatine kinase (CK), glycerate kinase, acetate kinase, glutamine synthetase, such as adenylated glutamine synthetase, and/or deadenylated glutamine synthetase, phosphofructokinase, and isoforms thereof. Instead, the method of the second, third, fourth or fifth aspect of the invention may be applied to a synthetase. The synthetase may then, for example, be glutamine synthetase (GC).

[0029] As also hereinbefore described, the deuterated imidazole moiety may be provided by a nucleotide or by a nucleotide analogue. The nucleotide may, in one embodiment of the invention be adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP), with the nucleotide having an immonium moiety at position N7, so that a carbene is induced at the C8 position, and with deuteration being effected at the C8 position. However, in another embodiment of the invention, the nucleotide may be a compound which is deuterated at a position equivalent to C8 in ATP, ADP and AMP.

[0030] The invention will now be described in more detail with reference to the accompanying non-limiting examples and drawings.

[0031] In the drawings

[0032] FIG. 1 shows, for Example 1, a protein sequence alignment of human adenylate kinase isoforms 1 to 6. KAD1=P00568 (SEQ ID NO. 1), KAD232 P54819 (SEQ ID NO. 2), KAD3=Q9UIJ7 (SEQ ID NO. 3), KAD4=P27144 (SEQ ID NO. 4), KAD5=Q9Y6K8 (SEQ ID NO. 5), and KAD6=Q9Y3D8 (SEQ ID NO. 6);

[0033] FIG. 2 shows, for Example 1, a protein sequence alignment of shikimate kinase isoforms 1 and 2: E. coli aroK, SK1_--Ecoli=P0A6D7 (SEQ ID NO. 7), E. coli aroL SK2_--Ecoli=P0A6E1 (SEQ ID NO. 8); Klebsiella pneumoniae aroK, SK1_--Kpueumoniae=A6TF14 (SEQ ID NO. 9), Klebsiella pneumoniae aroL, SK2_--Kpueumoniae=A6T5B3 (SEQ ID NO. 10); Yersinia pestis aroK, SK1_--Ypestis=A6BW25 (SEQ ID NO. 11), Yersinia pestis aroL, SK2ypestis=A4TPJ4 (SEQ ID NO. 12); Shigella flexneri aroK, SK1_--Sflexneri=Q0SZS8 (SEQ ID NO. 13), Shigella flexneri aroL, SK2_--Sflexneri=Q83M66 (SEQ ID NO. 14), and Mycobacterium tuberculosis aroK, SK1_--Mtuberculosis=P0A4Z2 (SEQ ID NO. 15);

[0034] FIG. 3 shows, for Example 1, a protein sequence alignment of pyruvate kinase isoforms R, L, M1 and M2. R=P12928 (SEQ ID NO. 16), L=P04763 (SEQ ID NO. 17), M1=P11980 (SEQ ID NO. 18) and M2=P11981 (SEQ ID NO. 19);

[0035] FIG. 4 shows, for Example 1, a protein sequence alignment of creatine kinase isoforms B-CK, cytoplasmic muscle M-CK, uMT and sMT: CKB=P12277 (SEQ ID NO. 20), CKM=P06732 (SEQ ID NO. 21), CKMT1A=P12532 (SEQ ID NO. 22), CKMT2=P17540 (SEQ ID NO. 23);

[0036] FIG. 5 shows, for Example 1, a protein sequence alignment of glycerate kinase isoforms 1; (SEQ ID NO. 24) and 2 (SEQ ID NO 25).;

[0037] FIG. 6 shows, for Example 1, a protein sequence alignment of human hexokinase isoforms 1-4 (SEQ ID NO. 26 to 29, respectively);

[0038] FIG. 7 shows, for Example 1, a protein sequence alignment of E. coli aspartokinase isoforms 1-3 (SEQ ID NO. 30 to 32, respectively);

[0039] FIG. 8 shows, for Example 1, conserved ATP binding domain sequence motifs in GSI-β compared to the domains in GS1-α and GSII: GLNA_SALTY=Salmonella typhimurium (P0A1 P6, SEQ ID NO 33), GLNA_THIFE=Acidithiobacillus ferrooxidans (P07804, SEQ ID NO 34), GLNA_--ECOLI=E. coli (P0A9C5; SEQ ID NO 35), GLNA_ARCFU=Archaeoglobus fulgidus ((O29380; SEQ ID NO 36), GLNA_AZOVI=Azotobacter vinelandii (P22248; SEQ ID NO 37), GLNA_Bacce=Bacillus cereus (P19064; SEQ ID NO 38), GLNA_BACSU=Bacillus subtilis (P12425; SEQ ID NO 39), GLNA_HALVO=Halobacterium volcanii (P43386; SEQ ID NO 40), GLNA_LACDE=Lactobacillus delbrueckii (P45627; SEQ ID NO 41)GLNA_PLASMO=Plasmodium falciparum (NCBI: XP001352097; SEQ ID NO 42), GLNA_YEAST=Saccharomyces cerevisiae (P32288; SEQ ID NO 43), GLNA_CHLRE-Chlamydomonas reinhardtii (Q42688; SEQ ID NO 44), GLNA_MAIZE-Zea mays (P49094; SEQ ID NO 45), GLNA_ORYSA=Orysa sativa (P14656; SEQ ID NO 46), GLNA_LUPLU-Lupinus luteus (P52782; SEQ ID NO 47), GLNA_PEA=Pisum sativum (P19251; SEQ ID NO 48), GLNA_DROME-Drosophila melanogaster (P20477; SEQ ID NO 49), GLNA_SQUAC=Squalus acanthia (P41520; SEQ ID NO 50), GLNA_XENLA =Xenopus laevis (P51121; SEQ ID NO 51), GLNA_CHICK=Gallus gallus (P16580; SEQ ID NO 52), GLNA_MOUSE=Mus musculus (P15105; SEQ ID NO 53), HAMSTR=Cricetulus griseus (PO4773; SEQ ID NO 54) and GLNA_HUMAN=Homo sapiens (P15104; SEQ ID NO 55).

[0040] FIG. 9 shows, for Example 1, stereo views of the interaction of identified Arg and Glu/Asp interaction in the active sites of in a range of kinase isoenzymes and a synthetase enzyme, namely AK (A), CK (B), PK (C), HXK, SK (D), ASK (E) and GS (F);

[0041] FIG. 10 shows, for Example 1, structure and numbering of ATP;

[0042] FIG. 11 shows, for Example 1, the role of Arg97 and Arg44 and the C8H of ATP in the binding of ATP and catalysing phosphoryl transfer in ATP dependent reactions;

[0043] FIG. 12 shows, for Example 2, the effect of the SDM R97K, R97Q, R97A, R128K, R128Q, R128A, E98L and D180L on the specific activity of AK1 with (A): R97K, R97Q, R128K, R128Q, and (B): R97A, R128A E98L and D180L;

[0044] FIG. 13 shows, for Example 3, the effect of pH and NaCl (.diamond-solid.), imidazole (.box-solid.), histidine (.tangle-solidup.) and 1,2 dimethyl imidazole (quadrature) on the enzyme activity of AK1. Activity is expressed as ADP produced in mM. Effect of pH on the protonation of imidazole (⋄), histidine (Δ) and 1,2 dimethyl imidazole ( );

[0045] FIG. 14 shows, for Example 4, the effect of imidazole.HCl and histidine.HCl and deuterated imidazole.HCl and histidine.HCl at a range of pH values on the activity of AK1, (.box-solid.) 2 mM NaCl, (.diamond-solid.) imidazole.HCl, (.tangle-solidup.) histidine.HCl, (⋄) deuterated imidazole.HCl and (quadrature) deuterated histidine.HCl;

[0046] FIG. 15 shows, for Example 5, the effect of the relative concentrations of ATP, AMP and C8-D ATP on the activity of AK1;

[0047] FIG. 16 shows, for Example 5, the effect of a range of concentrations of ATP (.diamond-solid.) and C8-D ATP (⋄) on the activity of AK1 and the KIE (.box-solid.);

[0048] FIG. 17 shows, for Example 6, the effect of Adenosine 5'-[y-thio]triphosphate (ATPS) (.diamond-solid.) and deuterated ATPS (⋄) on the synthesis of ADP by AK1. The activity of AK1 in the absence of ATPS produced 0.019 mM ADP; and

[0049] FIG. 18 shows, for Example 7, ¹H and ¹⁵N NMR HSQC spectrum of AK1 in the presence of ¹H-ATPS (light shading) and C8-D ATPS (darker shading). Arrows indicate shift changes as a result of the binding of the deuterated analogue.

[0050] FIG. 19 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of Mycobacteria tuberculosis shikimate kinase; =ATP, .box-solid.=C8D-ATP, a=KIE, b=KIE_D.

[0051] FIG. 20 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of Saccharomyces cerevisiae hexokinase; =ATP, .box-solid.=C8D-ATP a=KIE, b=KIE_D.

[0052] FIG. 21 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of Escherichia coli acetate kinase; =ATP, .box-solid.=C8D-ATP, a=KIE, b=KIE_D.

[0053] FIG. 22 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of Escherichia coli GS₀; =ATP, .box-solid.=C8D-ATP a=KIE, b=KIE_D.

[0054] FIG. 23 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of B. stearothermophilus phosphofructokinase; =ATP, .box-solid.=C8D-ATP, a=KIE, b=KIE_D.

[0055] FIG. 24 shows, for Example 8, the effect of the concentration of ATP and C8D-ATP on the specific activity (A) and KIE (B) of Escherichia coli adenylylated glutamine synthetase; =ATP, .box-solid.=C8D-ATP, a=KIE, b=KIE_D.

[0056] FIG. 25 shows, for Example 8, the effect of low concentrations of ATP on the KIE and KIE_D obtained for A: shikimate kinase, B: hexokinase, C: acetate kinase, D: GS₀, E: PFK and F: GS₁₂; a=KIE, b=KIE_D

[0057] FIG. 26 shows, for Example 8, models for the binding of nucleotides to kinases and synthetase enzymes. A: Model for the binding of ATP and release of ADP from monomeric kinase. The current model for oligomeric kinases is based on nonequivalent ligand binding where binding to one monomer affects binding to a second monomer, or coordinated active sites. B: Model based on the rate reaction in the conversion of ATP to ADP with the concomitant conversion of the second binding site to the ATP-binding-form as a result of the release of the ADP. Once the ADP has been released from the first site this changes the affinity of the second site from an ADP binding structure to an ATP binding structure. C: Model based on the rate reaction in the conversion of ATP to ADP with the concomitant conversion of the second binding site to the ATP-binding-form as a result of the conversion of ATP to ADP in the first binding site. Once the ATP is converted ADP this changes the affinity of the second site from an ADP-binding structure to an ATP-binding-form.

Example 1

[0058] The kinases are a large number of structurally diverse enzymes that play a critical role in numerous metabolic and signaling pathways and whose substrates may be a small molecule, lipid, or protein. The kinases have been classified into 25 families of homogenous proteins, with the families assembled into 12 fold-groups based on the similarity of their structural folds. The enzymes transferring high energy phosphate bonds from nucleotides into two divisions, namely, transferases (kinases) and ligases (synthetases). The catalytic and regulatory mechanisms employed in nucleotide binding and phosphoryl transfer, within a single kinase group from both prokaryotic and eukaryotic organisms, as well as the specific kinase isoenzymes, are kinetically and functionally distinct based on the rate of phosphoryl transfer and the regulation thereof. An initial structural comparison of key amino acid residues associated with phosphoryl transfer activity and the regulation of kinase enzymes and their isoenzymes, as depicted in FIGS. 1 to 8 and in Table 1, demonstrated the level of structural homology of the residues that are associated with phosphoryl transfer activity and/or the regulation of phosphoryl transfer activity, and whether functionality may be differentiated based on the conservation of key amino acids in the active sites of these kinases and the role of the conserved amino acid residues in phosphoryl transfer. The sequence and structural analysis of a range of kinase and synthetase enzymes was carried out using the Accelrys INSIGHTII and Discovery Studio 2.5 suite of molecular modeling software--see FIGS. 1 to 9. The protein sequence alignments of each enzymes isoforms were carried using the DNAMAN sequence alignment software using the dynamic alignment with a gap penalty of 10, a gap extension penalty of 5 and the BLOSUM protein weight.

[0059] Based on the structural analysis of the nucleotide binding site of a range of diverse kinase enzymes, as depicted in FIG. 9, the role of key amino acids involved in nucleotide binding was defined. The protein sequence alignments for each enzyme isoform and structural investigation of the active sites of adenylate kinase (AK), shikimate kinase (SK), pyruvate kinase (PK), hexokinase (HXK), aspartokinase (ASK) and creatine kinase (CK) indicated conserved paired Arg and Glu/Asp in the active site associated with the binding of the imidazole moiety and the α-PO₄ of the nucleotide (Table 1 and FIG. 9) (Sequence alignments: FIGS. 1-8, Structural comparison of conserved residues: FIG. 9 and Table 1). The AK group of enzymes isoforms served as a template for the identification of key amino acid residues that could be required for the binding and/or regulation of phosphoryl transfer within the active site of kinases. The mechanism of binding and the role of the imidazole moiety of the nucleotide in the binding and its role in the regulation of catalysis was defined. Within the active site of AK, SK, PK, HXK, ASK and CK, Arg and Glu/Asp residues associated with the binding ATP to the active site of the enzymes (Table 1), are conserved. The NH2 of the Arg97 is within a requisite 3.5 Å of the C-8 proton (ATP atom numbering: FIG. 10) of the ATP analogue. As the active sites of AK1 and AK4 were not distorted in the crystal structure, these sites were used to identify the imidazole binding domain within the active site. AK1 contains "diadenyl tetraphosphate" in the active site while AK4 contains "diadenyl pentaphosphate" in the active site. Hydrogen bonding occurs between conserved Arg and Glu or Asp acid residues and the imidazole moiety of the nucleotide. AK1 has the Arg 97 and Glu 98 and the pair Arg 128 and Asp 180 associated with the equivalent N7/C8 atoms of the diadenyl tetraphosphate. The equivalent residues in AK2-AK6 are outlined in Table 1. In CK, the Arg/Glu pair hydrogen bonded to the imidazole moiety of the nucleotide ATP are Arg 292 and Asp 335. Outlined in Table 1 and FIG. 9 are similar interactions between the C8-H of the imidazole moiety of the nucleotide and the conserved Arg residue within the active sites of AK, SK, PK, HXK, ASK, CK and GS isoenzymes. In all enzymes investigated, other than HXK I-III, a conserved Arg was identified within approximately 3.5 A of the C8-H of the imidazole moiety of the associated nucleotide. In HXK I-III, the Arg is replaced by a Lys whereas in HXK IV the Arg is conserved. The identified Arg residue was also found to be H-bonding distance of the α-PO₄ of the nucleotide. It is proposed the concomitant association of the Arg with the C8-H of the imidazole moiety and the α-PO₄ of the nucleotide has mechanistic implications in the regulation of the phosphoryl transfer activity of the enzyme. In all cases additional arginine residue/s are associated with the primary arginine residue which is associated with the imidazole moiety of the nucleotide. The second arginine residue is also associated with the phosphate backbone of the nucleotide. The arginine residues may be replaced by either glutamine or lysine.

[0060] It was therefore proposed that the C-8 proton of ATP plays a direct role in the binding of ATP to the active site as well as in the catalysis of phosphorylation by general acid-base catalysis as outlined in FIG. 11. The role of the Arg97 is to act as a general base catalyst in the abstraction of the C-8 proton from ATP which in turn, via resonance stabilization, acts to protonate the α-phosphate of the ATP. The second arginine in the active site, Arg 44, then acts to transfer the proton to the β-phosphate. This acid-catalysed stabilization of the α- and β-phosphates of the ATP acts to withdraw electrons into the "backbone" of the, α-β-γ-phosphate chain of ATP, allowing for resonance stabilization of the phosphate backbone and facilitating that the γ-phosphate acts as a better leaving group in the phosphorylation of the substrate molecules.

TABLE-US-00001 TABLE 1 Identified Arg and Glu/Asp residues responsible for the regulation of phosphoryl transfer in a range of kinase isoenzymes and a synthetase enzyme. The rows contain the conserved amino acid residues from various isoenzymes of each enzyme. Where structures contained a co-crystallized nucleotide the inter-atomic distances between the C8-H of the imidazole moiety, α-PO₄ of the nucleotide and the amino acid were measured (Atoms and inter-atomic distances outlined below each residue). Within each isoenzyme the Glu/Asp residues associated with each Arg residue follow the Arg residue in the column of the table. In PK, AK and CK the Arg is also in H-bonding distance of the C8-H of ATP as well the α-PO₄ of ATP. In SK1 a second Arg residue is responsible for the interaction with C8-H of ATP as well the α-PO₄ of ATP. PK- AK1^a AK5 AK6 AK2 AK3 AK4 M-CK uMt-CK sMt-CK PK-R PK-L M1/M2^a (Cyto) (Cyto) (Cyto) (Mito) (Mito) (Mito) (Cyto) (Mito) (Mito)^a His121 His89 His77 Arg97 Arg100 Arg? Arg103 Arg94 Arg92 Arg292 Arg325 Arg326 His ND₁ to ATP-C8H = 4.57 Å Arg NH₁ to ATP-α-PO₄ = 2.41 Å Arg NH₂ to ATP-C8H = 2.58 Å His NE2 to ATP-α-PO₄ = 1.92 Å Arg NH₂ to ATP-C8H = 3.46 Å Arg NH₂ to ATP-α-PO₄ = 3.58 Å His NE2 to Arg120 NH1 = 1.92 Å Arg HE to ATP-β-PO₄ = 1.67 Å Arg116 Arg84 Arg72 Glu98 Glu101 Asp77? Glu109 Glu100 Glu98? Arg236 Arg269 Arg270 Arg NH₁ to ATP-C8H = 4.60 Å Arg NH₁ to ATP-α-PO₄ = 3.15 Å Arg NH₁ to ATP-α-PO₄ = 1.92 Å Arg44 Arg47 Arg39 Arg51 Arg43 Arg41 Arg320 Arg352 Arg354 Arg NH₁ to ATP-α-PO₄ = 1.90 Å Arg NH₁ to ATP-α-PO₄ = 1.92 Å Arg NH₁ to ATP-α-PO₄ = 3.75 Å Asp335 Asp368 Asp369 Arg163 Arg132 Arg120 Arg128 Arg131 Arg105 Arg138 Arg124 Arg122 Asp δ-OD₁ to Arg326 NH₁ = 1.85 Å Arg NH₁ to ATP-β-PO₄ = 2.05 Å Arg NH₁ to ATP-C8H = 6.40 Å Asp180 Asp184 Asp157 Glu? Glu200 Glu198 SK1^a,b SK2^b HK1/HK2^a HK3 HK4 ASK1 ASK2 ASK3 GS Arg113 Arg NE Lys785^c Lys791 Arg333 Arg238 Arg241 Arg232 Arg 355 Arg HE to Glu108 δ-OD₁ = 2.39 Å Arg NH₁ to ATP-C8H = 4.33 Å Arg NH₂ to ATP-C8H = 3.95 Å Arg NH₁ to ATP-C8H = 4.00 Å Arg NH₂ to His271 ND1 = 3.33 Å Glu108 Glu NE Gln789 Gln NE Gln337 Arg306 Arg309 Arg300 Arg120 Arg117 Arg30^d Lys41 NE Glu 357 Arg NH₁ to ATP-α-PO₄ = 2.75 Å Arg NH₂ to ATP-C8H = 3.44 Å Asp228 Asp231 Asp222 Glu? NE Asp OD₁ to Arg30 NH₁ = 3.25 Å The PDB accession codes for protein structures used, pyruvate kinase (PK); 1A49, adenylate kinase (AK1); 2C95, creatine kinase (CK); 2GL6, Shikimate kinase (SK1), 1L4U, hexokinase (HXK1) catalytic site; 1DGK, hexokinase (HXK1) allosteric regulation site; 1QHA, glutamine synthetase (GS): 1F52. ^aIsoenzyme from which the associated inter-atomic distance data was obtained. ^bResidue number taken from E. coli sequence however Arg/Glu/Asp identification in structure taken from Mycobacteria tuberculosis structure as ADP is co-crystallized into the active site of this structure. ^cCatalytic subunit active site residue. ^dAllosteric regulation subunit binding site residue. NE = No equivalent residue in this isoenzyme

Example 2: Determination of the Role of Key Amino in AK1 Using Site-Directed Mutagenesis

[0061] The role of key amino acids involved in the nucleotide binding was demonstrated by site-directed mutagenesis of AK1 and enzymatic analysis of the mutated enzymes demonstrating the necessity of these amino acids in enzyme activity and nucleotide binding.

[0062] Construct AK1A-c001, encoding a N-terminal His-tagged human adenylate kinase 1 gene, was obtained from the Structural Genomics Consortium, University of Oxford, United Kingdom. Site-directed mutagenesis was carried out using Finnzymes' Phusion Site-Directed Mutagenesis kit. The mutations created were: Arg97 and Arg128 to Ala, Gln and Lys, and Glu98 and Asp180 to Leu. All resulting constructs were sequenced to confirm mutations. Wild-type and mutated versions of His-tagged AK1 were expressed in E. coli Origami(DE3) (Novagen), and purified using Bio-Rad's Profinia Purification System. Purified proteins were dialysed against 50 mM KH₂PO₄/K₂HPO₄ buffer (pH 6.8) containing 1.5 mM MgCl₂ and 120 mM KCl.

[0063] The effect of the SDM on the specific activity of AK1 was determined in assays containing 50 mM KH₂PO₄/K₂HPO₄, 0.6 mM ADP, 0.6 mM ATP and 0.66 mM MgCl₂ at 37° C. at pH 6.9. The reaction was stopped by the addition of trichloroacetic acid to a final pH of 2 to 3. The assay solutions were centrifuged prior to HPLC analysis. The assays for adenosine, AMP, ADP and ATP were carried out using a Phenomenex 5 μ LUNA C₁₈ column with the mobile phase containing PIC A® (Waters Corporation), 250 ml acetonitrile and 0.7% (w/v) KH₂PO₄. The flow rate of the mobile phase was 1 ml/min with UV detection.

[0064] The effect of the following SDM; R97K, R97Q, R97A, R128K, R128Q, R128A, E98L and D180L on the specific activity of AK1 was determined--see FIGS. 12A and B. In the case of both Arg97 and Arg128 mutations to Lys or Gln gave a significant decrease in the specific activity of the enzyme with the effect being 32 fold for R97K, 120 fold for R97Q, and 2200 fold in the case of R128K and R128Q. The R97A mutation gave a 100 fold reduction in activity while the R128A enzyme did not give detectable activity. The D180L mutation gave a 20 fold reduction in activity while the E98L mutation had no effect on the specific activity. The effect of the mutations on the Arg128/D180 pair was greater than the Arg97/Glu98 pair.

Example 3: Effect of pH on the Inhibition of AK1 by Imidazole.HCl, Histidine.HCl and 1,2 Dimethyl Imidazole.HCl

[0065] The effect of the enzyme assay pH and the presence of imidazole.HCl, histidine.HCl and 1,2 dimethyl imidazole.HCl on the activity of AK1 was determined. The assays contained 50 mM KH₂PO₄/K₂HPO₄ (at the equivalent pH), 0.6 mM ADP, 0.6 mM ATP, 0.66 mM MgCl₂ and either imidazole, histidine. or 1,2 dimethyl imidazole at a concentration of 2 mM.

[0066] The effect of imidazole.HCl, histidine.HCl and 1,2 dimethyl imidazole.HCl concentration on the activity of AK1 was determined and expressed as a relative activity, relative to the enzyme activity obtained in the presence of NaCl--see FIG. 13. The pH optimum for AK1 is of the order of pH 6.9. As each imidazole compound gave distinctive inhibition of the enzymes. The inhibition of AK1 appears to be related to the protonation of the imidazole moiety, with AK1 being inhibited by the deprotonated form of the imidazole--FIG. 13. As the concentration of the protonated imidazole in solution increases with decreasing pH there is a concomitant increase in the activity of the enzyme. At a pH above pH 6.9 the deprotonated imidazole acts an inhibitor of AK1.

Example 4: Effect of pH on the Inhibition of AK1 by Imidazole.HCl and Histidine.HCl and Deuterated Imidazole.HCl and Histidine.HCl.

[0067] The enzyme activity of AK1 was determined over a pH range of pH 6.3 to pH 8.1 in the presence of 2mM NaCl, imidazole.HCl and histidine.HCl and compared under the same conditions in the presence of deuterated imidazole.HCl and histidine.HCl--see FIG. 14. The imidazole.HCl and histidine.HCl was deuterated at position 5 which is equivalent to position C8 in the nucleotides. As outlined in Example 3, both imidazole.HCl and histidine.HCl inhibited the activity of AK1 when compared with the activity in the presence of NaCl. In both the case of imidazole.HCl and histidine.HCl there was a further reduction in the activity in excess of 50% of both adenylate kinases in the presence of deuterated imidazole.HCl and histidine.HCl.

[0068] Kinase inhibitors labeled at the carbon equivalent to C8 would therefore be at least double as inhibitory as unlabelled inhibitors.

Example 5: Effect of Deuterated AMP and ATP on the Activity of AK1.

[0069] The enzyme activity of AK1 was determined using undeuterated nucleotides and compared with the activity in the presence of deuterated AMP, deuterated ATP and deuterated AMP and ATP. The assays contained 50 mM KH₂PO₄/K₂HPO₄ (at the equivalent pH), 0.6 mM ADP, 0.6 mM ATP and 0.66 mM MgCl₂.

[0070] The enzyme activity of AK1 was determined using undeuterated nucleotides and compared with the activity in the presence of deuterated ATP and undeuterated ATP (FIGS. 15 and 16). At low ATP concentrations AK1 the presence of undeuterated ATP gave lower activity than when deuterated ATP was used. At high ATP concentrations there is at least a 50% reduction in the activity as is the case in the presence deuterated ATP when compared with deuterated ATP.

Example 6: The Effect of Adenosine 5'-[γ-thio]triphosphate (ATPS) and Deuterated ATPS on the Activity of AK1

[0071] The effect of ATPS and deuterated ATPS (C8-D ATPS) on the activity of AK1 was determined. Assays contained 1 nM human AK1, 0.66 mM MgCl₂, 0.6 mM ATP and 0.6 mM AMP in 50 mM potassium phosphate buffer, pH 7.2.

[0072] Increasing concentrations of ATPS or C8-D ATPS were added; 0.2, 0.4, 0.6, 0.8, and 1.0 mM. Assays, in a final volume of 1 ml, were allowed to proceed for 45 mins at 37° C., before 6 μl 100% TCA was added to stop the reaction. Both ATPS and C8-D ATPS were found to inhibit AK1; however the C8-D ATPS gave significantly more inhibition than the ATPS--see FIG. 17. The assay where no ATPS or C8-D ATPS was added produced 0.19 mM ADP.

Example 7

[0073] AK1 was labeled by culturing the E. coli expression cells in the presence of ¹⁵N NH₄Cl and purified. A Heteronuclear Single Quantum Coherence (HSQC) ¹H and ¹⁵N NMR spectrum was obtained of AK1 in the presence of ATPS and C8-D ATPS at a concentration of ≈=200 μM AK1 and 100 mM of ATPS or C8-D ATPS. From the HSQC spectrum it is clearly evident that a number of the shifts in the active site of AK1 change their relative position as a result of the binding of the nucleotide in either the ATPS or C8-D ATPS forms--see FIG. 18).

[0074] A non-classical kinetic isotope effect is thus found in the enzyme kinetics of AK1 at low ATP concentrations, with a 50% reduction in enzyme activity, when the reactions are carried out using ATP when compared with ATP deuterated at position C8. A 50% reduction in enzyme activity is also obtained in the case where the kinase and synthase enzymes perform reversible reactions employing ADP or AMP.

[0075] All nucleotides bind into the active site of adenylate kinase enzymes by this mechanism which forms part of catalysis by inducing of C8-H to become more acidic.

[0076] A number of "imidazole" moiety-containing compounds inhibit adenylate kinase enzymes. A further reduction in enzyme activity by at least 50% is obtained when the "imidazole" moiety-containing compounds are deuterated at the position equivalent to C8 in AMP/ADP/ATP.

[0077] In accordance with the invention, a coupling mechanism is therefore provided whereby nucleotides bind to the active site of enzymes at the imidazole moiety by the inter-conversion of the C8-H. This coupling mechanism also plays a role in imidazole inhibition of adenylate kinases.

[0078] "Imidazole" moiety-containing compounds mimic the imidazole moiety of nucleotides and bond transiently but covalently to the active site of adenylate kinase enzymes in the same manner as nucleotides do.

Example 8: The Effect of the ATP and C8D-ATP Concentration on the Specific Activity of Saccharomvces Cerevisiae Hexokinase, Escherichia Coli Acetate Kinase, Escherichia Coli Phosphofructokinase, Escherichia Coli Deadenylylated Glutamine Synthetase, Escherichia Coli Adenylylated Glutamine Synthetase and Mycobacterium Tuberculosis Shikimate Kinase

[0079] Based on the studies carried out on AK1 as presented in Examples 1 to 7, further comparative assays were run to determine the effect of ATP and C8D-ATP on the specific activity of a range of kinase enzymes. The kinase enzymes investigated were hexokinase, acetate kinase, shikimate kinase, phosphofructokinase, glutamine synthetase [(GS₁₂) and (GS₀)].

[0080] Acetate kinase (EC 2.7.2.1) is a homodimer which catalyses the Mg²+-dependent, reversible transfer of phosphate from ATP to acetate according to the following reaction:

CH₃COO^-+ATP⇄CH₃CO₂PO₃²-+ADP

[0081] Acetate kinase forms part of the acetate and sugar kinase/Hsc70/actin (ASKHA) structural superfamily (PFam Clan: Actin ATPase:CL0108). The enzyme is a homodimer, and monomer interaction plays a role in the regulation of the enzyme activity and ligand binding with the enzyme active sites functioning in a coordinated half-the-sites manner. The acting ATPase clan contains both the acetate kinases and sugar kinases, and are all known to undergo a catalytically essential domain closure upon ligand binding.

[0082] Hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) catalyses the Mg²+-dependent phosphorylation of glucose, from ATP:

C₆H₁₂O₆+ATP⇄C₆H₁₂O₆PO₃.su- p.2-+ADP

[0083] The two isoenzymes of yeast hexokinase, designated P-I and P-II, are dimers of subunit molecular mass 52 kDa. Hexokinase also forms part of the acetate and sugar structural superfamily (PFam Clan: Actin_ATPase:CL0108). Yeast hexokinase enzymes are structurally well characterised with each subunit of the homodimer comprising two domains and in the open conformation these domains are separated by a cleft containing the sugar binding sight. Binding of glucose induces a large conformational change in which the two lobes of the subunit rotate relative to each. The enzymes also exist in a monomer-dimer association-dissociation equilibrium that is influenced by pH, ionic strength and substrates. There are major differences in the glucose binding behaviour of both forms where binding to dimeric P-I shows strong positive cooperativity, whereas in P-II the two sites are equivalent and binding is non-cooperative.

[0084] Shikimate kinase. The shikimate pathway is a seven-step biosynthetic route that links the metabolism of carbohydrates to the synthesis of aromatic amino acids via the conversion of erythrose-4-phosphate to chorismic acid. Chorismic acid is an essential intermediate for the synthesis of aromatic compounds, such as aromatic amino acids, p-aminobenzoic acid, folate, and ubiquinone. Shikimate kinase (SK, EC 2.7.1.71), the fifth enzyme in the shikimate biosynthetic, catalyzes phosphate transfer from ATP to the carbon-3-hydroxyl group of shikimate, forming shikimate 3-phosphate. SK belongs to the nucleoside monophosphate (NMP) kinase structural family, with characteristic features of the NMP kinases being that they undergo large conformational changes during catalysis and belong to the P-loop containing nucleoside triphospahte hydrolase superfamily (Pfam Clan: AAA:CL0023). The NMP kinases are composed of three domains: the CORE, which contains a highly conserved phosphate-binding loop (P-loop); the LID domain, which undergoes substantial conformational changes upon substrate binding, and the NMP-binding domain, which is responsible for the recognition and binding of a specific substrate. MgADP induces concerted hinged movements of the shikimate binding and LID domains causing the two domains to move towards each other in the presence of this ligand. The SK crystal structures show that SK exists as a monomer with a single ATP binding site.

[0085] Phosphofructokinase (PFK, fructose-6-phosphate 1-kinase) (EC 2.7.1.11) is a classical allosteric enzyme that catalyzes the phosphorylation of D-fructose 6-phosphate (Fru-6-P) by Mg-ATP to form D-fructose 1,6-bisphosphate and MgADP. PFK from B. stearothermophilus is a homo-tetramer in which each subunit has a molecular weight of 34 000, and which undergoes a concerted two-state allosteric transition. PFK belongs to the PFK-like superfamily (Pfam Clan: PFK:CL0240) The enzyme from Bacillus stearothermophilus (Bs-PFK) shows hyperbolic Michaelis-Menten kinetics with respect to both Fru-6-P and Mg-ATP, but cooperative kinetics in the presence of allosteric inhibitor phosphoenolpyruvate(PEP). Unliganded Bs-PFK is in the active R state, which has high affinity for substrate, switching to the inactive T state with low affinity for substrate only in the presence of PEP.

[0086] Glutamine synthetase. Glutamine synthetase (GS) (EC 6.3.1.2) catalyzes the reversible conversion of L-glutamic acid, ATP and ammonia to L-glutamine, ADP and inorganic phosphate via a γ-glutamyl phosphate intermediate. As GS is a central enzyme in nitrogen metabolism the enzyme is regulated by at least four different mechanisms: (a) adenylylation and deadenylylation of the tyrosine 397 residue, (b) conversion between a relaxed (inactive) and taut (active) state depending on the divalent metal cation present, (c) cumulative feedback inhibition by multiple end products of glutamine metabolism, and (d) repression and derepression of GS biosynthesis in response to nitrogen availability. Escherichia coli GS is a large, metalloenzyme (˜624 kDa) comprising 12 identical subunits arranged in two face-to-face hexagonal rings. E. coli GS belongs to the glutamine synthetase 1-β group of enzymes that are regulated via adenylylation of a single tyrosine residue, with each subunit requiring two structurally implicated divalent cations (either Mg²+ or Mn²+) for its catalytic activity. The extent of adenylylation of the E. coli GS in response to an excess or deficiency of nitrogen in the growth environment is regulated in response to the intracellular concentrations of 2-ketoglutarate and glutamine, via the reversible adenylylation of a tyrosine residue (Tyr397) in each subunit of GS. The presence of adenylylated GS (GS₁₂) predominates in a nitrogen-rich, carbon-limited media, while the deadenylylated form (GS₀) tends to predominate under conditions of nitrogen limitation. The regulation of the adenylylation state of GS is accomplished by three proteins, uridylyltransferase/uridylyl-removing enzyme, the signal transduction protein P_II, and adenylyltransferase. High intracellular concentrations of glutamine activate the uridylyl-removing enzyme which causes the deuridylylation of P_II. This interacts with the adenylyltransferase which then catalyses the adenylylation of the GS. High intracellular concentrations of 2-ketoglutarate activate uridylyltransferase, which transfers UMP to each subunit of P_II, forming P_II-UMP. The P_II-UMP interacts with the adenylyltransferase, which in turn catalyses the removal of AMP from the GS.

[0087] Materials and Methods

[0088] Enzyme Source, and Protein Expression and Purification

[0089] Hexokinase from Saccharomyces cerevisiae Type F-300 (Sigma, H4502) and Acetate kinase from E. coli (Sigma, A7437) were purchased. The human adenylate kinase (AK1) gene in vector pLIG-SC1 was obtained from the Structural Genomics Consortium (code AK1A). The His-tagged AK1 was produced in Escherichia coli Origami (DE3) and purified using the Bio-Rad Profinia Purification System. The pure protein was dialysed against 50 mM KH₂PO₄/K₂HPO₄ buffer (pH 6.8), 1.5 mM MgCl₂, 120 mM KCl. The Mycobacterium tuberculosis shikimate kinase gene in pET15b (Novagen) was obtained from the group of Chris Abell, Cambridge University, UK. The his-tagged MtSK was produced in E. coli BL21 (DE3) and purified using the Bio-Rad Profinia Purification System. The pure protein was dialysed against 50 mM Tris (pH 7.5) and 1,000 mM NaCl. Adenylylated (GS₁₂) and deadenylylated (GS₀) glutamine synthetase were prepared as outlined below.

[0090] Production of glnD and glnE Knockout Strains

[0091] Knockout strains for the production of fully adenylylated (glnD knockout) or fully deadenylylated GS (glnE knockout) were made from the E. coli YMC11 using the Quick & Easy E. coli Gene Deletion Kit (Gene Bridges GmbH), designed to knockout or alter genes on the E. coli chromosome. Red/ET recombination allows the exchange of genetic information in a base pair precise and specific manner. An FRT-flanked kanamycin resistance marker cassette is supplied with the kit which can be used to replace a gene on the E. coli chromosome. The use of a FRT-flanked resistance cassette for the replacement of the targeted gene allows the subsequent removal of the selection marker by a FLP-recombinase step, involving the transformation of an FLP-expression plasmid into the cells and subsequent expression of an FLP site-specific recombinase. The genes for the Recombination proteins are under the control of an inducible promoter and the plasmid carries a temperature sensitive origin of replication for a convenient removal of the plasmid after recombination. In order to produce fully adenylylated GS, it is necessary to knockout the uridylyltransferase, coded by the glnD gene.

[0092] Primers were designed to the E. coli glnD gene. These primers contained a region specific to the glnD gene adjoining a sequence specific to the FRT cassette (underlined, see below). In a similar fashion, to produce fully deadenylylated GS, the adenylyltrasnferase, coded by the glnE gene, needs to be knocked out. Primers were therefore designed to the E. coli glnE gene. These primers contained a region specific to the glnE gene adjoining a sequence specific to the FRT cassette (underlined, see below). Both knockout strains were produced using the primers as described in the kit protocol. The only deviation from the protocol, was that BamHI restriction sites were incorporated in the ends of the primers (shown in bold). This enabled the PCR product to be cloned into pGEM T-Easy (Promega Corporation), and then cut out of the pGEM construct as a BamHI fragment. This facilitated production of the cassette in sufficient quantity for the transformation step, as it was found to be extremely difficult to produce enough of the cassette by PCR alone. Once integration of the cassette was confirmed by selection on kanamycin plates, a PCR product was produced using primers designed to the sequence of the glnD or glnE gene, either side of the integration site. This PCR product was then sequenced to confirm integration. The kanamycin resistance marker was removed using the 706-FLP plasmid carrying the site-specific recombinase. The removal of the marker was also confirmed by sequencing, as above. Primers used to create glnD and glnE knockout strains of E. coli YMC11 were:

TABLE-US-00002 glnD sense primer, (SEQ ID NO. 56) 5'-gaggatcccagaaccagcgccatcagcgttaccatggcaccagc tacaaccttgaaccaattaaccctcactaaagggcg-3'; glnD antisense primer, (SEQ ID NO. 57) 5'-gtggatccgcgatatcgtgaaacagcgcggcgatgaaaatcagc tcagttgacggcagtaatacgactcactatagggctc-3'; glnE sense primer, (SEQ ID NO. 58) 5'-gaggatcctgcgcctgtttgaactgacgcagcgcctcaagctgt tgctcttcgtcatcaattaaccctcactaaagggcg-3'; glnE antisense primer, (SEQ ID NO. 59) 5'-gtggatccaggtgttccagctcattcgcggcggacgcgaaccgt cgctgcaatcgcgctaatacgactcactatagggctc-3'.

[0093] Purification of E. Coli Glutamine Synthetase

[0094] GS₁₂ and GS₀ were purified from recombinant E. coli YMC11 glnD and glnE knockout strains. E. coli YMC11 glnD strain producing GS₁₂ and the E. coli YMC11 glnE strain producing GS₀. The culturing protocols used were as outlined in supplementary information. The enzyme concentration and purity were determined by Quant -IT® Protein Assay Kit (Invitrogen, USA) and SDS-PAGE.

[0095] C8-D ATP Synthesis.

[0096] The synthesis ATP and ADP deuterated at the C8 position (C8-D ATP and C8-D ADP) was carried out based on the method of (49). A 20 mM solution of Na₂ATP in D₂O containing 60 mM triethylamine (TEA) was incubated at 60° C. for 144 hours. The TEA was removed by twice passing the solution over a Dowex 20 W ion-exchange resin in the acid form. The pH of the solution was adjusted to pH 12 with NaOH prior to the second pass over the resin. The pH of the solution was adjusted to pH 6.3 prior to freeze drying. The extent of the deuteration of the C8 proton was determined by ¹H NMR and mass spectroscopy.

[0097] Steady-State Kinetic Analysis

[0098] GS₁₂, and GS₀ assay. The effect of the concentration of ATP and C8D-ATP on the specific activity of GS₁₂, and GS₀ was determined at concentrations ranging from 150 to 3000 μM ATP and C8D-ATP in assays containing 4 mM Na-glutamate, 4 mM NH₄Cl, 5.4 mM NaHCO₃ in 20 mM imidazole buffer. The GS₀ assay was carried out at pH 7.4 (±pH 0.05), and at MgCl₂ concentrations equivalent to 3 times the ATP concentration. The GS₁₂ assay was carried out at pH 6.6 (±pH 0.05), and at MnCl₂ concentrations equivalent to 3 times the ATP concentration. The reaction was stopped by the addition of tri-chloroacetic acid to give a pH of 2-3. The forward reaction rate was determined by measuring the ADP concentration in solution HPLC.

[0099] Hexokinase assay: 100 mM Phosphate buffer pH 6.8, 10 mM D-Glucose, 250 mM KCl, MgCl₂ and ATP were kept at a 1:1 ratio at concentration between 0.2 mM-3 mM. The assay was incubated at 37° C. for 15 minutes and stopped by the addition of 1 μl of 50% TCA. The formation of ADP was analysed by HPLC.

[0100] Acetatekinase assay: 100 mM Phosphate buffer pH 6.8, 10 mM Sodium Acetate, 250 mM KCl, MgCl₂ and ATP were kept at a 1:1 ratio at concentration between 0.2 mM-3 mM. The assay was incubated at 30° C. for 30 minutes and stopped by the addition of 1 μl of 50% TCA. The formation of ADP was analysed by HPLC.

[0101] Phosphofructokinase assay: 100 mM Phosphate buffer pH 6.8, 10 mM Fructose-6-Phosphate, 250 mM KCl, MgCl₂ and ATP were kept at a 1:1 ratio at concentration between 0.2 mM-3 mM. The assay was incubated at 37° C. for 15-30 minutes and stopped by the addition of 1 μl of 50% TCA. The formation of ADP was analysed by HPLC.

[0102] Shikimate kinase assay: Assays comprised 100 mM potassium phosphate buffer (pH 6.8), 500 mM KCl, 10 nM enzyme, and varying amounts of ATP, shikimic acid and MgCl₂. These were kept at a constant ratio of 1:1:2 for ATP: MgCl₂: shikimic acid. The ATP concentrations ranged between 0.2 and 10 mM. The final volumes were 100 μl, and the reactions were incubated at 37° C. for 20 minutes, before being terminated by the addition of 5 μl 200 mM EDTA.

[0103] In all assays, the production of ADP was analysed by HPLC.The assay solutions were centrifuged prior to HPLC analysis. The assays for adenosine, AMP, ADP ATP were carried out using Phenomenex 5p LUNA C₁₈ column with the mobile phase containing PIC A® (Waters Corporation), 250 ml acetonitrile, 7 g KH₂PO₄ per litre water. The flow rate of the mobile phase was 1 ml/minute with UV detection.

[0104] Results

[0105] ATP was deuterated specifically at position C8 as hereinbefore described and the deuteration was assessed by ¹H NMR.

[0106] The effect of the ATP and C8D-ATP concentration on the specific activity of Saccharomyces cerevisiae hexokinase, Escherichia coli acetate kinase, Escherichia coli phosphofructokinase, Escherichia coli deadenylylated glutamine synthetase, Escherichia coli adenylylated glutamine synthetase and Mycobacterium tuberculosis shikimate kinase was determined. The results are reflected in FIGS. 19 to 25. Where possible, the effect of the ATP and C8D-ATP on the specific activity of the enzyme was expressed over a concentration profile that included the ATP or C8D-ATP concentrations that would allow v_max to be calculated as well as an ATP or C8D-ATP concentration profile at low concentrations that would allow for the accurate determination of the KIE. The best-fit to the data was obtained for the specified kinetic model using the non-linear regression algorithms as outlined using the GraphPad Prism® 5 software. As part of the software output, a data-table was created containing 150 data-points defining the best kinetic fit for each enzymes response to the presence of either ATP or C8D-ATP (see table 2 for kinetic model). These response curves were then used to define the KIE by the conventional estimation of KIE from KIE=v_H/v_D. The KIE_D was also determined using the following function:

KIE D = v D v H ##EQU00001## [0107] Where v_D=specific activity in the presence of C8D-ATP [0108] v_H=specific activity in the presence of ATP.

[0109] The calculation of KIE_D was used as the data obtained is instructive in a putative role that the C8H of ATP plays in the regulation of phosphoryl transfer.

[0110] In all 6 cases defined: [0111] A KIE was obtained in response to presence of C8D-ATP (FIGS. 19-24). [0112] In all cases other than shikimate kinase, the KIE_D at low ATP concentrations is in excess of 5 (FIG. 25). [0113] In monomeric enzymes, such as shikimate kinase, as the concentration of ATP and C8D-ATP was increased, there was a concomitant increase in the KIE_D while in oligomeric enzymes a there was a decrease in the KIE_D (FIG. 19). [0114] In all cases the KIE obtained was a primary KIE as extent of the KIE was two-fold or significantly in excess of two-fold at low concentrations. [0115] The KIE_D over the full ATP/C8D-ATP concentration range appeared to be indicative of the mode of regulation of the enzyme as in all cases the KIE either positively or negatively asymptotes to a specific constant value. [0116] The KIE of shikimate kinase asymptotes positively to a KIE of 1.0 as the specific activity tends towards v_max. The KIE giving a classical KIE effect with the KIE being 2 at low ATP concentrations asymptoting to a level of 1 (FIG. 19, Table 2). Shikimate kinase exists as a monomer and therefore no regulation occurs via the interaction of the subunits that may affect the overall KIE. [0117] Hexokinase, acetate kinase and GS₀ use the same mechanism for regulation. The KIE_D of these enzymes negatively asymptote to 1 at V_max (FIGS. 20-22, Table 2). All three of these enzymes are multi-meric and allosteric regulation may occur via the interaction of sub-units. The hexokinase and acetate kinase are both homodimers and monomer interaction plays a role in the regulation of the enzyme activity and ligand binding with the enzyme active sites functioning in a coordinated half-the-sites manner. [0118] Phosphofructokinase and GS₁₂ use a similar mechanism with the KIE_D asymptoting to a level of 0.5 at v_max (KIE=2) (FIGS. 22-24, Table 2). E. coli GS₁₂ is a dodecamer consisting of two stacked hexameric structures consisting of 12 identical subunits. The subunits probably interact allosterically on the binding of ATP as occurs in phosphofructokinase. The slow rate of release of C8D-ADP from the interacting active site of GS₁₂ probably impacting on the binding of ATP in the adjacent site.

TABLE-US-00003 [0118] TABLE 2 Effect of the concentration of ATP and C8D-ATP on the fit of the enzyme kinetic model of hexokinase, acetate kinase, adenylylated GS, deadenylylated GS and shikimate kinase. The response of each enzyme to change in the ATP and C8D-ATP concentration was tested for the fit to either an allosteric sigmoidal model or to the Michaelis-Menton model of enzyme kinetics by non-linear regression using the GraphPrism 5 software. The root mean square deviation of the data from the model is as outlined. The Hill factor for the allosteric sigmoidal model is as indicated. KIE_vmax is equal to the KIE attained at ATP and C8D-ATP concentrations at maximum enzyme activities. ATP fit to kinetic C8D-ATP fit to RMSD^a Enzyme Kinetic Model model kinetic model h_ATP h.sub.C8D ATP C8D KIE_vmax Hexokinase Allosteric sigmoidal Allosteric sigmoidal Fit ambiguous 1.75 0.9963 1 Michaelis-Menton Fit ambiguous Michaelis-Menton 0.9885 Acetate kinase Allosteric sigmoidal Allosteric sigmoidal Fit ambiguous 2.08 0.9865 1 Michaelis-Menton Fit ambiguous Michaelis-Menton 0.9847 Dedenylylated GS Allosteric sigmoidal Allosteric sigmoidal Allosteric sigmoidal 3.10 4.08 0.9972 0.9827 1 Michaelis-Menton Fit ambiguous Fit ambiguous PFK^b Allosteric sigmoidal Allosteric sigmoidal Allosteric sigmoidal 1.37 1.79 0.9982 0.9879 2 Michaelis-Menton Fit ambiguous Fit ambiguous Adenylylated GS Allosteric sigmoidal Allosteric sigmoidal Allosteric sigmoidal 1.77 0.92 0.9940 0.9990 2 Michaelis-Menton Fit ambiguous Fit ambiguous Shikimate kinase Allosteric sigmoidal Allosteric sigmoidal Allosteric sigmoidal 1.22 1.06 0.9870 0.9770 1 Michaelis-Menton Fit ambiguous Fit ambiguous ^aRoot mean square deviation of the data defining the kinetic model. ^bPhosphofructokinase

[0119] Discussion

[0120] The role of the KIE in the kinetics of the enzymes investigated lead to models for the regulation of the binding of ATP being proposed, as set out in FIG. 25.

[0121] In classical steady-state kinetics as represented by the Briggs-Haldane modification of the Michael-Menton formulation (Equation 1),

E + S  → k on k off ES → k cat E + P ( Eq . 1 ) ##EQU00002##

and k_on=k₁, k_off=k_-1 and k_cat=k₂, k₂>>k_-1, and the Michaelis constant, K_M is obtained from

[ E ] [ S ] [ ES ] = k - 1 + k 2 k 1 = K M ( Eq . 2 ) ##EQU00003##

[0122] In monomeric enzymes such a shikimate kinase K_M is dependent only on k₂. The effect of the increase in the ATP/C8D-ATP concentration on the KIE therefore only manifests as the classical effect with the KIE being of the order of 2.0 as determined by v_H/v_D, at low concentrations, asymptoting to 1 at high ATP concentrations. At low concentrations of ATP the enzyme activity is dominated by the impact of the C8H/C8D on the equilibrium of binding. At high ATP concentrations the impact of the increase in the ATP concentration on the equilibrium overrides the effect of the C8H/C8D on binding resulting in a decrease in the KIE. As the classical H/D KIE is of the order of 2, as the concentration of ATP tends towards the concentration at the maximum specific activity, v_max, where the concentration effect is at its maximum the effect of the C8H/C8D on the KIE is at a minimum and the KIE tends towards 1.

[0123] In oligomeric enzymes it is proposed that the deuteration of ATP not only affects the binding of ATP to the site where catalysis is occurring but the deuteration also affects the interaction between sites. In oligomeric kinases it is proposed that mechanistically two modes of regulation occur, one which is dependent on the release of ADP from the first active site before ATP binds to the second active site (FIG. 26B) and the second mode of regulation depends on the conversion of ATP to ADP prior to the binding of the ATP to the second active site (FIG. 26C). In the mechanism outlined in FIG. 25C binding to the second site can occur prior to the release of ATP from the first site.

[0124] It is proposed that in enzymes such as acetate kinase, hexokinase and GS₁₂ the enzyme kinetics follows classical Michaelis-Menton kinetics where an equilibrium is set up between the enzyme concentration [E] and the substrate concentration [S] and binding of the second ATP is dependent on the conversion of the second active site into an ATP binding form by the release of ATP from the first active site, as defined by the coordinated half-sites mechanism. In enzymes using this mechanism of regulation, K_M is dependent on k_-1 and k₂. The KIE obtained in these enzymes asymptotes to a value of 1. At low ATP concentrations the effect of the deuteration of C8 is to allow binding to occur for long enough to allow the reaction to occur and negate the effect of k_-1, thereby shifting the equilibrium to k₂. At low ATP concentrations therefore the impact of the deuteration on the binding is to retard the release of the ATP. At high ATP concentrations the impact of the ATP concentration relative to the impact of ATP binding on the rate of reaction is significantly higher and as a result there is a concomitant increase in the KIE. The impact of binding and the reaction rate however equilibrate to a KIE of 1. The maximum rate of binding can only ever be equivalent to the maximum rate at which the second ATP binding site is converted to the ATP binding form by the release of ATP from the first site (FIG. 26B). The classical impact of deuteration on the KIE when the KIE is a primary effect, as determined by v_H/v_D, should yield a KIE of 2 or more. As the regulation of the enzyme activity and ligand binding in these enzymes function in a coordinated half-the-sites manner binding in the second site only occurs on release of the ADP from the first site, it is therefore proposed that deuteration of the ATP improves the binding characteristics. As the equilibrium shifts towards the impact of increasing ATP concentration on the enzyme activity the deuterated ATP binds effectively twice as efficiently as the non-deuterated ATP thereby negating the impact of the deuteration on the apparent enzyme activity at high ATP concentrations, yielding a KIE of 1.

[0125] In enzymes where the second active site is made amenable to ATP binding by the conversion of ATP to ADP, in other words binding may occur to the second site prior to the release of the ATP from the first site, the K_M is dependent on k₁ and k₂. This occurs in the case of phosphofructokinase and GS₁₂ where the KIE becomes 2 at v_max (FIGS. 23 & 24). The impact of this binding is that at any point in time up to two or more reactions might be occurring simultaneously in two active sites. In multi-meric enzymes this effect might be greater. As the deuterated ATP binds twice as efficiently as the non-deuterated ATP this allows the KIE to asymptote to 2 or more. It is proposed that a result of the adenylylation of GS it allows for the regulation of the enzyme by a similar mechanism as occurs in phosphofructokinase. Bacterial PFK is a homoteramer, with the four subunits assembled as a dimer of dimers. It is conceivable that on adenylylation of GS the interaction between two-subunits effectively creates a dimer of dimer interaction.

[0126] The data outlined clearly demonstrates the role of C8H of ATP in the kinetics of a number of kinase and synthetase enzymes. The KIE is clearly a primary KIE however the extremely high values of the KIE obtained at low at concentrations in the case of the oligomeric enzymes does not appear to be as a result of the impact of the deuterium on the rate the phosphoryl transfer mechanism per se but rather as a result of the role that the C8H plays in the equilibrium of binding of the ATP to the active site (FIG. 25). Clearly the regulation of enzyme activity in kinases and synthetases is complex which manifests in the apparent K_m of the kinases ranges from less than 0.4 μM to in excess of 1000 μM for ATP (Carna Biosciences, Inc., Kinase Profiling Book:www.carnabio.com). The findings of this investigation have discovered that the C8H of ATP plays a direct role in binding of ATP to the active site of enzymes.

[0127] The deuteration of compounds containing imidazole moieties that are currently used as drugs, will increase their efficacy. With the increase in the efficacy of the deuterated forms of current drugs containing imidazole moieties either in use or in clinical trails, dosage levels of these compounds may be reduced to alleviate the toxicity.

Sequence CWU 1

591194PRTHomo sapiens 1Met Glu Glu Lys Leu Lys Lys Thr Lys Ile Ile Phe Val Val Gly Gly1 5 10 15Pro Gly Ser Gly Lys Gly Thr Gln Cys Glu Lys Ile Val Gln Lys Tyr 20 25 30Gly Tyr Thr His Leu Ser Thr Gly Asp Leu Leu Arg Ser Glu Val Ser 35 40 45Ser Gly Ser Ala Arg Gly Lys Lys Leu Ser Glu Ile Met Glu Lys Gly 50 55 60Gln Leu Val Pro Leu Glu Thr Val Leu Asp Met Leu Arg Asp Ala Met65 70 75 80Val Ala Lys Val Asn Thr Ser Lys Gly Phe Leu Ile Asp Gly Tyr Pro 85 90 95Arg Glu Val Gln Gln Gly Glu Glu Phe Glu Arg Arg Ile Gly Gln Pro 100 105 110Thr Leu Leu Leu Tyr Val Asp Ala Gly Pro Glu Thr Met Thr Gln Arg 115 120 125Leu Leu Lys Arg Gly Glu Thr Ser Gly Arg Val Asp Asp Asn Glu Glu 130 135 140Thr Ile Lys Lys Arg Leu Glu Thr Tyr Tyr Lys Ala Thr Glu Pro Val145 150 155 160Ile Ala Phe Tyr Glu Lys Arg Gly Ile Val Arg Lys Val Asn Ala Glu 165 170 175Gly Ser Val Asp Ser Val Phe Ser Gln Val Cys Thr His Leu Asp Ala 180 185 190Leu Lys2239PRTHomo sapiens 2Met Ala Pro Ser Val Pro Ala Ala Glu Pro Glu Tyr Pro Lys Gly Ile1 5 10 15Arg Ala Val Leu Leu Gly Pro Pro Gly Ala Gly Lys Gly Thr Gln Ala 20 25 30Pro Arg Leu Ala Glu Asn Phe Cys Val Cys His Leu Ala Thr Gly Asp 35 40 45Met Leu Arg Ala Met Val Ala Ser Gly Ser Glu Leu Gly Lys Lys Leu 50 55 60Lys Ala Thr Met Asp Ala Gly Lys Leu Val Ser Asp Glu Met Val Val65 70 75 80Glu Leu Ile Glu Lys Asn Leu Glu Thr Pro Leu Cys Lys Asn Gly Phe 85 90 95Leu Leu Asp Gly Phe Pro Arg Thr Val Arg Gln Ala Glu Met Leu Asp 100 105 110Asp Leu Met Glu Lys Arg Lys Glu Lys Leu Asp Ser Val Ile Glu Phe 115 120 125Ser Ile Pro Asp Ser Leu Leu Ile Arg Arg Ile Thr Gly Arg Leu Ile 130 135 140His Pro Lys Ser Gly Arg Ser Tyr His Glu Glu Phe Asn Pro Pro Lys145 150 155 160Glu Pro Met Lys Asp Asp Ile Thr Gly Glu Pro Leu Ile Arg Arg Ser 165 170 175Asp Asp Asn Glu Lys Ala Leu Lys Ile Arg Leu Gln Ala Tyr His Thr 180 185 190Gln Thr Thr Pro Leu Ile Glu Tyr Tyr Arg Lys Arg Gly Ile His Ser 195 200 205Ala Ile Asp Ala Ser Gln Thr Pro Asp Val Val Phe Ala Ser Ile Leu 210 215 220Ala Ala Phe Ser Lys Ala Thr Cys Lys Asp Leu Val Met Phe Ile225 230 2353227PRTHomo sapiens 3Met Gly Ala Ser Ala Arg Leu Leu Arg Ala Val Ile Met Gly Ala Pro1 5 10 15Gly Ser Gly Lys Gly Thr Val Ser Ser Arg Ile Thr Thr His Phe Glu 20 25 30Leu Lys His Leu Ser Ser Gly Asp Leu Leu Arg Asp Asn Met Leu Arg 35 40 45Gly Thr Glu Ile Gly Val Leu Ala Lys Ala Phe Ile Asp Gln Gly Lys 50 55 60Leu Ile Pro Asp Asp Val Met Thr Arg Leu Ala Leu His Glu Leu Lys65 70 75 80Asn Leu Thr Gln Tyr Ser Trp Leu Leu Asp Gly Phe Pro Arg Thr Leu 85 90 95Pro Gln Ala Glu Ala Leu Asp Arg Ala Tyr Gln Ile Asp Thr Val Ile 100 105 110Asn Leu Asn Val Pro Phe Glu Val Ile Lys Gln Arg Leu Thr Ala Arg 115 120 125Trp Ile His Pro Ala Ser Gly Arg Val Tyr Asn Ile Glu Phe Asn Pro 130 135 140Pro Lys Thr Val Gly Ile Asp Asp Leu Thr Gly Glu Pro Leu Ile Gln145 150 155 160Arg Glu Asp Asp Lys Pro Glu Thr Val Ile Lys Arg Leu Lys Ala Tyr 165 170 175Glu Asp Gln Thr Lys Pro Val Leu Glu Tyr Tyr Gln Lys Lys Gly Val 180 185 190Leu Glu Thr Phe Ser Gly Thr Glu Thr Asn Lys Ile Trp Pro Tyr Val 195 200 205Tyr Ala Phe Leu Gln Thr Lys Val Pro Gln Arg Ser Gln Lys Ala Ser 210 215 220Val Thr Pro2254223PRTHomo sapiens 4Met Ala Ser Lys Leu Leu Arg Ala Val Ile Leu Gly Pro Pro Gly Ser1 5 10 15Gly Lys Gly Thr Val Cys Gln Arg Ile Ala Gln Asn Phe Gly Leu Gln 20 25 30His Leu Ser Ser Gly His Phe Leu Arg Glu Asn Ile Lys Ala Ser Thr 35 40 45Glu Val Gly Glu Met Ala Lys Gln Tyr Ile Glu Lys Ser Leu Leu Val 50 55 60Pro Asp His Val Ile Thr Arg Leu Met Met Ser Glu Leu Glu Asn Arg65 70 75 80Arg Gly Gln His Trp Leu Leu Asp Gly Phe Pro Arg Thr Leu Gly Gln 85 90 95Ala Glu Ala Leu Asp Lys Ile Cys Glu Val Asp Leu Val Ile Ser Leu 100 105 110Asn Ile Pro Phe Glu Thr Leu Lys Asp Arg Leu Ser Arg Arg Trp Ile 115 120 125His Pro Pro Ser Gly Arg Val Tyr Asn Leu Asp Phe Asn Pro Pro His 130 135 140Val His Gly Ile Asp Asp Val Thr Gly Glu Pro Leu Val Gln Gln Glu145 150 155 160Asp Asp Lys Pro Glu Ala Val Ala Ala Arg Leu Arg Gln Tyr Lys Asp 165 170 175Val Ala Lys Pro Val Ile Glu Leu Tyr Lys Ser Arg Gly Val Leu His 180 185 190Gln Phe Ser Gly Thr Glu Thr Asn Lys Ile Trp Pro Tyr Val Tyr Thr 195 200 205Leu Phe Ser Asn Lys Ile Thr Pro Ile Gln Ser Lys Glu Ala Tyr 210 215 2205198PRTHomo sapiens 5Met Gly Gly Phe Met Glu Asp Leu Arg Lys Cys Lys Ile Ile Phe Ile1 5 10 15Ile Gly Gly Pro Gly Ser Gly Lys Gly Thr Gln Cys Glu Lys Leu Val 20 25 30Glu Lys Tyr Gly Phe Thr His Leu Ser Thr Gly Glu Leu Leu Arg Glu 35 40 45Glu Leu Ala Ser Glu Ser Glu Arg Ser Lys Leu Ile Arg Asp Ile Met 50 55 60Glu Arg Gly Asp Leu Val Pro Ser Gly Ile Val Leu Glu Leu Leu Lys65 70 75 80Glu Ala Met Val Ala Ser Leu Gly Asp Thr Arg Gly Phe Leu Ile Asp 85 90 95Gly Tyr Pro Arg Glu Val Lys Gln Gly Glu Glu Phe Gly Arg Arg Ile 100 105 110Gly Asp Pro Gln Leu Val Ile Cys Met Asp Cys Ser Ala Asp Thr Met 115 120 125Thr Asn Arg Leu Leu Gln Arg Ser Arg Ser Ser Leu Pro Val Asp Asp 130 135 140Thr Thr Lys Thr Ile Ala Lys Arg Leu Glu Ala Tyr Tyr Arg Ala Ser145 150 155 160Ile Pro Val Ile Ala Tyr Tyr Glu Thr Lys Thr Gln Leu His Lys Ile 165 170 175Asn Ala Glu Gly Thr Pro Glu Asp Val Phe Leu Gln Leu Cys Thr Ala 180 185 190Ile Asp Ser Ile Ile Phe 1956172PRTHomo sapiens 6Met Leu Leu Pro Asn Ile Leu Leu Thr Gly Thr Pro Gly Val Gly Lys1 5 10 15Thr Thr Leu Gly Lys Glu Leu Ala Ser Lys Ser Gly Leu Lys Tyr Ile 20 25 30Asn Val Gly Asp Leu Ala Arg Glu Glu Gln Leu Tyr Asp Gly Tyr Asp 35 40 45Glu Glu Tyr Asp Cys Pro Ile Leu Asp Glu Asp Arg Val Val Asp Glu 50 55 60Leu Asp Asn Gln Met Arg Glu Gly Gly Val Ile Val Asp Tyr His Gly65 70 75 80Cys Asp Phe Phe Pro Glu Arg Trp Phe His Ile Val Phe Val Leu Arg 85 90 95Thr Asp Thr Asn Val Leu Tyr Glu Arg Leu Glu Thr Arg Gly Tyr Asn 100 105 110Glu Lys Lys Leu Thr Asp Asn Ile Gln Cys Glu Ile Phe Gln Val Leu 115 120 125Tyr Glu Glu Ala Thr Ala Ser Tyr Lys Glu Glu Ile Val His Gln Leu 130 135 140Pro Ser Asn Lys Pro Glu Glu Leu Glu Asn Asn Val Asp Gln Ile Leu145 150 155 160Lys Trp Ile Glu Gln Trp Ile Lys Asp His Asn Ser 165 1707173PRTEscherichia coli 7Met Ala Glu Lys Arg Asn Ile Phe Leu Val Gly Pro Met Gly Ala Gly1 5 10 15Lys Ser Thr Ile Gly Arg Gln Leu Ala Gln Gln Leu Asn Met Glu Phe 20 25 30Tyr Asp Ser Asp Gln Glu Ile Glu Lys Arg Thr Gly Ala Asp Val Gly 35 40 45Trp Val Phe Asp Leu Glu Gly Glu Glu Gly Phe Arg Asp Arg Glu Glu 50 55 60Lys Val Ile Asn Glu Leu Thr Glu Lys Gln Gly Ile Val Leu Ala Thr65 70 75 80Gly Gly Gly Ser Val Lys Ser Arg Glu Thr Arg Asn Arg Leu Ser Ala 85 90 95Arg Gly Val Val Val Tyr Leu Glu Thr Thr Ile Glu Lys Gln Leu Ala 100 105 110Arg Thr Gln Arg Asp Lys Lys Arg Pro Leu Leu His Val Glu Thr Pro 115 120 125Pro Arg Glu Val Leu Glu Ala Leu Ala Asn Glu Arg Asn Pro Leu Tyr 130 135 140Glu Glu Ile Ala Asp Val Thr Ile Arg Thr Asp Asp Gln Ser Ala Lys145 150 155 160Val Val Ala Asn Gln Ile Ile His Met Leu Glu Ser Asn 165 1708174PRTEscherichia coli 8Met Thr Gln Pro Leu Phe Leu Ile Gly Pro Arg Gly Cys Gly Lys Thr1 5 10 15Thr Val Gly Met Ala Leu Ala Asp Ser Leu Asn Arg Arg Phe Val Asp 20 25 30Thr Asp Gln Trp Leu Gln Ser Gln Leu Asn Met Thr Val Ala Glu Ile 35 40 45Val Glu Arg Glu Glu Trp Ala Gly Phe Arg Ala Arg Glu Thr Ala Ala 50 55 60Leu Glu Ala Val Thr Ala Pro Ser Thr Val Ile Ala Thr Gly Gly Gly65 70 75 80Ile Ile Leu Thr Glu Phe Asn Arg His Phe Met Gln Asn Asn Gly Ile 85 90 95Val Val Tyr Leu Cys Ala Pro Val Ser Val Leu Val Asn Arg Leu Gln 100 105 110Ala Ala Pro Glu Glu Asp Leu Arg Pro Thr Leu Thr Gly Lys Pro Leu 115 120 125Ser Glu Glu Val Gln Glu Val Leu Glu Glu Arg Asp Ala Leu Tyr Arg 130 135 140Glu Val Ala His Ile Ile Ile Asp Ala Thr Asn Glu Pro Ser Gln Val145 150 155 160Ile Ser Glu Ile Arg Ser Ala Leu Ala Gln Thr Ile Asn Cys 165 1709173PRTKlebsiella pneumoniae 9Met Ala Glu Lys Arg Asn Ile Phe Leu Val Gly Pro Met Gly Ala Gly1 5 10 15Lys Ser Thr Ile Gly Arg Gln Leu Ala Gln Gln Leu Asn Met Glu Phe 20 25 30Tyr Asp Ser Asp Gln Glu Ile Glu Lys Arg Thr Gly Ala Asp Val Gly 35 40 45Trp Val Phe Asp Val Glu Gly Glu Glu Gly Phe Arg Asp Arg Glu Glu 50 55 60Lys Ile Ile Asn Glu Leu Thr Glu Lys Gln Gly Ile Val Leu Ala Thr65 70 75 80Gly Gly Gly Ser Val Lys Ser Arg Glu Thr Arg Asn Arg Leu Ser Ala 85 90 95Arg Gly Val Val Val Tyr Leu Glu Thr Thr Ile Glu Lys Gln Leu Ala 100 105 110Arg Thr Gln Arg Asp Lys Lys Arg Pro Leu Leu Gln Val Asp Ala Pro 115 120 125Pro Arg Glu Val Leu Glu Ala Leu Ala Asp Glu Arg Asn Pro Leu Tyr 130 135 140Glu Glu Ile Ala Asp Val Thr Ile Arg Thr Asp Asp Gln Ser Ala Lys145 150 155 160Val Val Ala Asn Gln Ile Ile His Met Leu Glu Ser Asn 165 17010177PRTKlebsiella pneumoniae 10Met Thr Gln Pro Ile Phe Leu Ile Gly Pro Arg Gly Cys Gly Lys Thr1 5 10 15Thr Val Gly His Ala Leu Ala Arg Ala Arg His Phe Gln Phe Ser Asp 20 25 30Thr Asp His Arg Leu Gln Ala His Glu Gln Arg Thr Val Ala Glu Ile 35 40 45Val Gln Ala Glu Gly Trp Ala Arg Phe Arg Glu Leu Glu Thr Leu Ser 50 55 60Leu Lys Ala Val Thr Leu Pro Asn Thr Val Ile Ala Thr Gly Gly Gly65 70 75 80Ile Val Leu Ala Glu Gly Asn Arg Gln Phe Met Arg Glu Asn Gly Val 85 90 95Val Ile Tyr Leu Gln Ala Ser Val Ser Ala Leu Ile Asp Arg Leu Glu 100 105 110Ala Tyr Pro Lys Ala Glu Gln Arg Pro Thr Leu Thr Gly Lys Pro Val 115 120 125Arg Glu Glu Val Gly Glu Val Leu Ala Gln Arg Glu Ala Leu Tyr Arg 130 135 140Asp Ala Ala His His Ile Val Asp Ala Thr Ala Ser Pro Asp Arg Val145 150 155 160Val Glu Gln Ile Met Ser Met Leu Cys Ser Ala Thr Ala Thr Pro Val 165 170 175Ser11173PRTYersinia pestis 11Met Ala Glu Lys Arg Asn Ile Phe Leu Val Gly Pro Met Gly Ala Gly1 5 10 15Lys Ser Thr Ile Gly Arg Gln Leu Ala Gln Gln Leu Asn Met Glu Phe 20 25 30Phe Asp Ser Asp Gln Glu Ile Glu Arg Arg Thr Gly Ala Asp Val Gly 35 40 45Trp Val Phe Asp Val Glu Gly Glu Glu Gly Phe Arg Asp Arg Glu Glu 50 55 60Lys Val Ile Asn Glu Leu Thr Glu Lys Gln Gly Ile Val Leu Ala Thr65 70 75 80Gly Gly Gly Ser Val Lys Ser Arg Glu Thr Arg Asn Arg Leu Ser Ala 85 90 95Arg Gly Val Val Val Tyr Leu Glu Thr Thr Ile Glu Lys Gln Leu Ala 100 105 110Arg Thr Gln Arg Asp Lys Lys Arg Pro Leu Leu Gln Val Asp Glu Pro 115 120 125Pro Arg Glu Val Leu Glu Ala Leu Ala Lys Glu Arg Asn Pro Leu Tyr 130 135 140Glu Glu Ile Ala Asp Val Thr Ile Arg Thr Asp Asp Gln Ser Ala Lys145 150 155 160Val Val Ala Asn Gln Ile Ile Asn Met Leu Glu Ser Asn 165 17012174PRTYersinia pestis 12Met Thr Gln Thr Ile Phe Met Val Gly Ala Arg Gly Ala Gly Lys Thr1 5 10 15Thr Ile Gly Lys Ala Leu Ala Gln Ala Leu Gly Tyr Arg Phe Val Asp 20 25 30Thr Asp Leu Phe Met Gln Gln Thr Ser Gln Met Thr Val Ala Glu Val 35 40 45Val Glu Ser Glu Gly Trp Asp Gly Phe Arg Leu Arg Glu Ser Met Ala 50 55 60Leu Gln Ala Val Thr Ala Pro Lys Thr Val Val Ala Thr Gly Gly Gly65 70 75 80Ala Val Leu Ser Ser Glu Asn Arg Ala Phe Met Arg Asp His Gly Arg 85 90 95Val Ile Tyr Leu Arg Ala Ser Ala Ala Val Leu Ala Lys Arg Leu Ala 100 105 110Glu Asp Pro Glu Glu Ala Gln Arg Pro Ser Leu Thr Gly Lys Pro Ile 115 120 125Val Glu Glu Ile Leu Asp Val Leu Ala Ser Arg Glu Ala Leu Tyr Gln 130 135 140Asp Val Ala His His Val Leu Asp Gly Thr Gln Thr Pro Ser Leu Val145 150 155 160Val Glu Gln Ile Leu Gln Met Leu Thr Gly Glu Met Val Lys 165 17013173PRTShigella flexneri 13Met Ala Glu Lys Arg Asn Ile Phe Leu Val Gly Pro Met Gly Ala Gly1 5 10 15Lys Ser Thr Ile Gly Arg Gln Leu Ala Gln Gln Leu Asn Met Glu Phe 20 25 30Tyr Asp Ser Asp Gln Glu Ile Glu Lys Arg Thr Gly Ala Asp Val Gly 35 40 45Trp Val Phe Asp Leu Glu Gly Glu Glu Gly Phe Arg Asp Arg Glu Glu 50 55 60Lys Val Ile Asn Glu Leu Thr Glu Lys Gln Gly Ile Val Leu Ala Thr65 70 75 80Gly Gly Gly Ser Val Lys Ser Arg Glu Thr Arg Asn Arg Leu Ser Ala 85 90 95Arg Gly Val Val Val Tyr Leu Glu Thr Thr Ile Glu Lys Gln Leu Ala 100 105 110Arg Thr Gln Arg Asp Lys Lys Arg Pro Leu Leu His Val Glu Thr Pro 115 120 125Pro Arg Glu Val Leu Glu Ala Leu Ala Asn Glu Arg Asn Pro Leu Tyr 130 135 140Glu Glu Ile Ala Asp Val Thr Ile Arg Thr Asp Asp Gln Ser Ala Lys145

150 155 160Val Val Ala Asn Gln Ile Ile His Met Leu Glu Ser Asn 165 17014174PRTShigella flexneri 14Met Thr Gln Pro Leu Phe Leu Ile Gly Pro Arg Gly Cys Gly Lys Thr1 5 10 15Thr Val Gly Met Ala Leu Ala Asp Ser Leu Asn Arg Arg Phe Val Asp 20 25 30Thr Asp Gln Trp Leu Gln Ser Gln Leu Asn Met Thr Val Ala Glu Ile 35 40 45Val Glu Arg Glu Glu Trp Ala Gly Phe Arg Ala Arg Glu Thr Ala Ala 50 55 60Leu Glu Ala Val Thr Ala Ala Ser Thr Val Ile Ala Thr Gly Gly Gly65 70 75 80Ile Ile Leu Thr Glu Phe Asn Arg His Phe Met Gln Asn Asn Gly Ile 85 90 95Val Val Tyr Leu Cys Ala Pro Val Ser Val Leu Val Asn Arg Leu Gln 100 105 110Ala Ala Pro Glu Glu Asp Leu Arg Pro Thr Leu Thr Gly Lys Pro Leu 115 120 125Ser Glu Glu Val Gln Glu Val Leu Glu Glu Arg Asp Ala Leu Tyr Arg 130 135 140Glu Val Ala His Ile Ile Ile Asp Ala Thr Asn Glu Pro Ser Gln Val145 150 155 160Ile Ser Glu Ile Arg Ser Ala Leu Ala Gln Thr Ile Asn Cys 165 17015176PRTMycobacterium tuberculosis 15Met Ala Pro Lys Ala Val Leu Val Gly Leu Pro Gly Ser Gly Lys Ser1 5 10 15Thr Ile Gly Arg Arg Leu Ala Lys Ala Leu Gly Val Gly Leu Leu Asp 20 25 30Thr Asp Val Ala Ile Glu Gln Arg Thr Gly Arg Ser Ile Ala Asp Ile 35 40 45Phe Ala Thr Asp Gly Glu Gln Glu Phe Arg Arg Ile Glu Glu Asp Val 50 55 60Val Arg Ala Ala Leu Ala Asp His Asp Gly Val Leu Ser Leu Gly Gly65 70 75 80Gly Ala Val Thr Ser Pro Gly Val Arg Ala Ala Leu Ala Gly His Thr 85 90 95Val Val Tyr Leu Glu Ile Ser Ala Ala Glu Gly Val Arg Arg Thr Gly 100 105 110Gly Asn Thr Val Arg Pro Leu Leu Ala Gly Pro Asp Arg Ala Glu Lys 115 120 125Tyr Arg Ala Leu Met Ala Lys Arg Ala Pro Leu Tyr Arg Arg Val Ala 130 135 140Thr Met Arg Val Asp Thr Asn Arg Arg Asn Pro Gly Ala Val Val Arg145 150 155 160His Ile Leu Ser Arg Leu Gln Val Pro Ser Pro Ser Glu Ala Ala Thr 165 170 17516574PRTRattus Norvegicus 16Met Ser Val Gln Glu Asn Thr Leu Pro Gln Gln Leu Trp Pro Trp Ile1 5 10 15Phe Arg Ser Gln Lys Asp Leu Ala Lys Ser Ala Leu Ser Gly Ala Pro 20 25 30Gly Gly Pro Ala Gly Tyr Leu Arg Arg Ala Ser Val Ala Gln Leu Thr 35 40 45Gln Glu Leu Gly Thr Ala Phe Phe Gln Gln Gln Gln Leu Pro Ala Ala 50 55 60Met Ala Asp Thr Phe Leu Glu His Leu Cys Leu Leu Asp Ile Asp Ser65 70 75 80Gln Pro Val Ala Ala Arg Ser Thr Ser Ile Ile Ala Thr Ile Gly Pro 85 90 95Ala Ser Arg Ser Val Asp Arg Leu Lys Glu Met Ile Lys Ala Gly Met 100 105 110Asn Ile Ala Arg Leu Asn Phe Ser His Gly Ser His Glu Tyr His Ala 115 120 125Glu Ser Ile Ala Asn Ile Arg Glu Ala Thr Glu Ser Phe Ala Thr Ser 130 135 140Pro Leu Ser Tyr Arg Pro Val Ala Ile Ala Leu Asp Thr Lys Gly Pro145 150 155 160Glu Ile Arg Thr Gly Val Leu Gln Gly Gly Pro Glu Ser Glu Val Glu 165 170 175Ile Val Lys Gly Ser Gln Val Leu Val Thr Val Asp Pro Lys Phe Gln 180 185 190Thr Arg Gly Asp Ala Lys Thr Val Trp Val Asp Tyr His Asn Ile Thr 195 200 205Arg Val Val Ala Val Gly Gly Arg Ile Tyr Ile Asp Asp Gly Leu Ile 210 215 220Ser Leu Val Val Gln Lys Ile Gly Pro Glu Gly Leu Val Thr Glu Val225 230 235 240Glu His Gly Gly Ile Leu Gly Ser Arg Lys Gly Val Asn Leu Pro Asn 245 250 255Thr Glu Val Asp Leu Pro Gly Leu Ser Glu Gln Asp Leu Leu Asp Leu 260 265 270Arg Phe Gly Val Gln His Asn Val Asp Ile Ile Phe Ala Ser Phe Val 275 280 285Arg Lys Ala Ser Asp Val Leu Ala Val Arg Asp Ala Leu Gly Pro Glu 290 295 300Gly Gln Asn Ile Lys Ile Ile Ser Lys Ile Glu Asn His Glu Gly Val305 310 315 320Lys Lys Phe Asp Glu Ile Leu Glu Val Ser Asp Gly Ile Met Val Ala 325 330 335Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu Lys Val Phe Leu Ala 340 345 350Gln Lys Met Met Ile Gly Arg Cys Asn Leu Ala Gly Lys Pro Val Val 355 360 365Cys Ala Thr Gln Met Leu Glu Ser Met Ile Thr Lys Ala Arg Pro Thr 370 375 380Arg Ala Glu Thr Ser Asp Val Ala Asn Ala Val Leu Asp Gly Ala Asp385 390 395 400Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly Ser Phe Pro Val Glu 405 410 415Ala Val Met Met Gln His Ala Ile Ala Arg Glu Ala Glu Ala Ala Val 420 425 430Tyr His Arg Gln Leu Phe Glu Glu Leu Arg Arg Ala Ala Pro Leu Ser 435 440 445Arg Asp Pro Thr Glu Val Thr Ala Ile Gly Ala Val Glu Ala Ser Phe 450 455 460Lys Cys Cys Ala Ala Ala Ile Ile Val Leu Thr Lys Thr Gly Arg Ser465 470 475 480Ala Gln Leu Leu Ser Gln Tyr Arg Pro Arg Ala Ala Val Ile Ala Val 485 490 495Thr Arg Ser Ala Gln Ala Ala Arg Gln Val His Leu Ser Arg Gly Val 500 505 510Phe Pro Leu Leu Tyr Arg Glu Pro Pro Glu Ala Ile Trp Ala Asp Asp 515 520 525Val Asp Arg Arg Val Gln Phe Gly Ile Glu Ser Gly Lys Leu Arg Gly 530 535 540Phe Leu Arg Val Gly Asp Leu Val Ile Val Val Thr Gly Trp Arg Pro545 550 555 560Gly Ser Gly Tyr Thr Asn Ile Met Arg Val Leu Ser Val Ser 565 57017543PRTRattus norvegicus 17Met Glu Gly Pro Ala Gly Tyr Leu Arg Arg Ala Ser Val Ala Gln Leu1 5 10 15Thr Gln Glu Leu Gly Thr Ala Phe Phe Gln Gln Gln Gln Leu Pro Ala 20 25 30Ala Met Ala Asp Thr Phe Leu Glu His Leu Cys Leu Leu Asp Ile Asp 35 40 45Ser Gln Pro Val Ala Ala Arg Ser Thr Ser Ile Ile Ala Thr Ile Gly 50 55 60Pro Ala Ser Arg Ser Val Asp Arg Leu Lys Glu Met Ile Lys Ala Gly65 70 75 80Met Asn Ile Ala Arg Leu Asn Phe Ser His Gly Ser His Glu Tyr His 85 90 95Ala Glu Ser Ile Ala Asn Ile Arg Glu Ala Thr Glu Ser Phe Ala Thr 100 105 110Ser Pro Leu Ser Tyr Arg Pro Val Ala Ile Ala Leu Asp Thr Lys Gly 115 120 125Pro Glu Ile Arg Thr Gly Val Leu Gln Gly Gly Pro Glu Ser Glu Val 130 135 140Glu Ile Val Lys Gly Ser Gln Val Leu Val Thr Val Asp Pro Lys Phe145 150 155 160Gln Thr Arg Gly Asp Ala Lys Thr Val Trp Val Asp Tyr His Asn Ile 165 170 175Thr Arg Val Val Ala Val Gly Gly Arg Ile Tyr Ile Asp Asp Gly Leu 180 185 190Ile Ser Leu Val Val Gln Lys Ile Gly Pro Glu Gly Leu Val Thr Glu 195 200 205Val Glu His Gly Gly Ile Leu Gly Ser Arg Lys Gly Val Asn Leu Pro 210 215 220Asn Thr Glu Val Asp Leu Pro Gly Leu Ser Glu Gln Asp Leu Leu Asp225 230 235 240Leu Arg Phe Gly Val Gln His Asn Val Asp Ile Ile Phe Ala Ser Phe 245 250 255Val Arg Lys Ala Ser Asp Val Leu Ala Val Arg Asp Ala Leu Gly Pro 260 265 270Glu Gly Gln Asn Ile Lys Ile Ile Ser Lys Ile Glu Asn His Glu Gly 275 280 285Val Lys Lys Phe Asp Glu Ile Leu Glu Val Ser Asp Gly Ile Met Val 290 295 300Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu Lys Val Phe Leu305 310 315 320Ala Gln Lys Met Met Ile Gly Arg Cys Asn Leu Ala Gly Lys Pro Val 325 330 335Val Cys Ala Thr Gln Met Leu Glu Ser Met Ile Thr Lys Ala Arg Pro 340 345 350Thr Arg Ala Glu Thr Ser Asp Val Ala Asn Ala Val Leu Asp Gly Ala 355 360 365Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly Ser Phe Pro Val 370 375 380Glu Ala Val Met Met Gln His Ala Ile Ala Arg Glu Ala Glu Ala Ala385 390 395 400Val Tyr His Arg Gln Leu Phe Glu Glu Leu Arg Arg Ala Ala Pro Leu 405 410 415Ser Arg Asp Pro Thr Glu Val Thr Ala Ile Gly Ala Val Glu Ala Ser 420 425 430Phe Lys Cys Cys Ala Ala Ala Ile Ile Val Leu Thr Lys Thr Gly Arg 435 440 445Ser Ala Gln Leu Leu Ser Gln Tyr Arg Pro Arg Ala Ala Val Ile Ala 450 455 460Val Thr Arg Ser Ala Gln Ala Ala Arg Gln Val His Leu Ser Arg Gly465 470 475 480Val Phe Pro Leu Leu Tyr Arg Glu Pro Pro Glu Ala Ile Trp Ala Asp 485 490 495Asp Val Asp Arg Arg Val Gln Phe Gly Ile Glu Ser Gly Lys Leu Arg 500 505 510Gly Phe Leu Arg Val Gly Asp Leu Val Ile Val Val Thr Gly Trp Arg 515 520 525Pro Gly Ser Gly Tyr Thr Asn Ile Met Arg Val Leu Ser Val Ser 530 535 54018531PRTRattus norvegicus 18Met Ser Lys Ser His Ser Glu Ala Gly Ser Ala Phe Ile Gln Thr Gln1 5 10 15Gln Leu His Ala Ala Met Ala Asp Thr Phe Leu Glu His Met Cys Arg 20 25 30Leu Asp Ile Asp Ser Ala Pro Ile Thr Ala Arg Asn Thr Gly Ile Ile 35 40 45Cys Thr Ile Gly Pro Ala Ser Arg Ser Val Glu Thr Leu Lys Glu Met 50 55 60Ile Lys Ser Gly Met Asn Val Ala Arg Met Asn Phe Ser His Gly Thr65 70 75 80His Glu Tyr His Ala Glu Thr Ile Lys Asn Val Arg Thr Ala Thr Glu 85 90 95Ser Phe Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu 100 105 110Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly Leu Ile Lys Gly Ser Gly 115 120 125Thr Ala Glu Val Glu Leu Lys Lys Gly Ala Thr Leu Lys Ile Thr Leu 130 135 140Asp Asn Ala Tyr Met Glu Lys Cys Asp Glu Asn Ile Leu Trp Leu Asp145 150 155 160Tyr Lys Asn Ile Cys Lys Val Val Asp Val Gly Ser Lys Val Tyr Val 165 170 175Asp Asp Gly Leu Ile Ser Leu Gln Val Lys Gln Lys Gly Pro Asp Phe 180 185 190Leu Val Thr Glu Val Glu Asn Gly Gly Phe Leu Gly Ser Lys Lys Gly 195 200 205Val Asn Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu Lys 210 215 220Asp Ile Gln Asp Leu Lys Phe Gly Val Glu Gln Asp Val Asp Met Val225 230 235 240Phe Ala Ser Phe Ile Arg Lys Ala Ala Asp Val His Glu Val Arg Lys 245 250 255Ile Leu Gly Glu Lys Gly Lys Asn Ile Lys Ile Ile Ser Lys Ile Glu 260 265 270Asn His Glu Gly Val Arg Arg Phe Asp Glu Ile Leu Glu Ala Ser Asp 275 280 285Gly Ile Met Val Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu 290 295 300Lys Val Phe Leu Ala Gln Lys Met Ile Ile Gly Arg Cys Asn Arg Ala305 310 315 320Gly Lys Pro Val Ile Cys Ala Thr Gln Met Leu Glu Ser Met Ile Lys 325 330 335Lys Pro Arg Pro Thr Arg Ala Glu Gly Ser Asp Val Ala Asn Ala Val 340 345 350Leu Asp Gly Ala Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly 355 360 365Asp Tyr Pro Leu Glu Ala Val Arg Met Gln His Leu Ile Ala Arg Glu 370 375 380Ala Glu Ala Ala Met Phe His Arg Lys Leu Phe Glu Glu Leu Ala Arg385 390 395 400Ser Ser Ser His Ser Thr Asp Leu Met Glu Ala Met Ala Met Gly Ser 405 410 415Val Glu Ala Ser Tyr Lys Cys Leu Ala Ala Ala Leu Ile Val Leu Thr 420 425 430Glu Ser Gly Arg Ser Ala His Gln Val Ala Arg Tyr Arg Pro Arg Ala 435 440 445Pro Ile Ile Ala Val Thr Arg Asn His Gln Thr Ala Arg Gln Ala His 450 455 460Leu Tyr Arg Gly Ile Phe Pro Val Val Cys Lys Asp Pro Val Gln Glu465 470 475 480Ala Trp Ala Glu Asp Val Asp Leu Arg Val Asn Leu Ala Met Asn Val 485 490 495Gly Lys Ala Arg Gly Phe Phe Lys Lys Gly Asp Val Val Ile Val Leu 500 505 510Thr Gly Trp Arg Pro Gly Ser Gly Phe Thr Asn Thr Met Arg Val Val 515 520 525Pro Val Pro 53019531PRTRattus norvegicus 19Met Pro Lys Pro Asp Ser Glu Ala Gly Thr Ala Phe Ile Gln Thr Gln1 5 10 15Gln Leu His Ala Ala Met Ala Asp Thr Phe Leu Glu His Met Cys Arg 20 25 30Leu Asp Ile Asp Ser Ala Pro Ile Thr Ala Arg Asn Thr Gly Ile Ile 35 40 45Cys Thr Ile Gly Pro Ala Ser Arg Ser Val Glu Met Leu Lys Glu Met 50 55 60Ile Lys Ser Gly Met Asn Val Ala Arg Leu Asn Phe Ser His Gly Thr65 70 75 80His Glu Tyr His Ala Glu Thr Ile Lys Asn Val Arg Ala Ala Thr Glu 85 90 95Ser Phe Ala Ser Asp Pro Ile Leu Tyr Arg Pro Val Ala Val Ala Leu 100 105 110Asp Thr Lys Gly Pro Glu Ile Arg Thr Gly Leu Ile Lys Gly Ser Gly 115 120 125Thr Ala Glu Val Glu Leu Lys Lys Gly Ala Thr Leu Lys Ile Thr Leu 130 135 140Asp Asn Ala Tyr Met Glu Lys Cys Asp Glu Asn Ile Leu Trp Leu Asp145 150 155 160Tyr Lys Asn Ile Cys Lys Val Val Glu Val Gly Ser Lys Ile Tyr Val 165 170 175Asp Asp Gly Leu Ile Ser Leu Gln Val Lys Glu Lys Gly Ala Asp Tyr 180 185 190Leu Val Thr Glu Val Glu Asn Gly Gly Ser Leu Gly Ser Lys Lys Gly 195 200 205Val Asn Leu Pro Gly Ala Ala Val Asp Leu Pro Ala Val Ser Glu Lys 210 215 220Asp Ile Gln Asp Leu Lys Phe Gly Val Glu Gln Asp Val Asp Met Val225 230 235 240Phe Ala Ser Phe Ile Arg Lys Ala Ala Asp Val His Glu Val Arg Lys 245 250 255Val Leu Gly Glu Lys Gly Lys Asn Ile Lys Ile Ile Ser Lys Ile Glu 260 265 270Asn His Glu Gly Val Arg Arg Phe Asp Glu Ile Leu Glu Ala Ser Asp 275 280 285Gly Ile Met Val Ala Arg Gly Asp Leu Gly Ile Glu Ile Pro Ala Glu 290 295 300Lys Val Phe Leu Ala Gln Lys Met Met Ile Gly Arg Cys Asn Arg Ala305 310 315 320Gly Lys Pro Val Ile Cys Ala Thr Gln Met Leu Glu Ser Met Ile Lys 325 330 335Lys Pro Arg Pro Thr Arg Ala Glu Gly Ser Asp Val Ala Asn Ala Val 340 345 350Leu Asp Gly Ala Asp Cys Ile Met Leu Ser Gly Glu Thr Ala Lys Gly 355 360 365Asp Tyr Pro Leu Glu Ala Val Arg Met Gln His Leu Ile Ala Arg Glu 370 375 380Ala Glu Ala Ala Val Phe His Arg Leu Leu Phe Glu Glu Leu Ala Arg385 390 395 400Ala Ser Ser Gln Ser Thr Asp Pro Leu Glu Ala Met Ala Met Gly Ser 405 410 415Val Glu Ala Ser Tyr Lys Cys Leu Ala Ala Ala Leu Ile Val Leu Thr 420 425 430Glu Ser Gly Arg Ser Ala His Gln Val Ala

Arg Tyr Arg Pro Arg Ala 435 440 445Pro Ile Ile Ala Val Thr Arg Asn Pro Gln Thr Ala Arg Gln Ala His 450 455 460Leu Tyr Arg Gly Ile Phe Pro Val Leu Cys Lys Asp Ala Val Leu Asp465 470 475 480Ala Trp Ala Glu Asp Val Asp Leu Arg Val Asn Leu Ala Met Asn Val485 490 495Gly Lys Ala Arg Gly Phe Phe Lys Lys Gly Asp Val Val Ile Val Leu 500 505 510Thr Gly Trp Arg Pro Gly Ser Gly Phe Thr Asn Thr Met Arg Val Val 515 520 525Pro Val Pro 53020381PRTHomo sapiens 20Met Pro Phe Ser Asn Ser His Asn Ala Leu Lys Leu Arg Phe Pro Ala1 5 10 15Glu Asp Glu Phe Pro Asp Leu Ser Ala His Asn Asn His Met Ala Lys 20 25 30Val Leu Thr Pro Glu Leu Tyr Ala Glu Leu Arg Ala Lys Ser Thr Pro 35 40 45Ser Gly Phe Thr Leu Asp Asp Val Ile Gln Thr Gly Val Asp Asn Pro 50 55 60Gly His Pro Tyr Ile Met Thr Val Gly Cys Val Ala Gly Asp Glu Glu65 70 75 80Ser Tyr Glu Val Phe Lys Asp Leu Phe Asp Pro Ile Ile Glu Asp Arg 85 90 95His Gly Gly Tyr Lys Pro Ser Asp Glu His Lys Thr Asp Leu Asn Pro 100 105 110Asp Asn Leu Gln Gly Gly Asp Asp Leu Asp Pro Asn Tyr Val Leu Ser 115 120 125Ser Arg Val Arg Thr Gly Arg Ser Ile Arg Gly Phe Cys Leu Pro Pro 130 135 140His Cys Ser Arg Gly Glu Arg Arg Ala Ile Glu Lys Leu Ala Val Glu145 150 155 160Ala Leu Ser Ser Leu Asp Gly Asp Leu Ala Gly Arg Tyr Tyr Ala Leu 165 170 175Lys Ser Met Thr Glu Ala Glu Gln Gln Gln Leu Ile Asp Asp His Phe 180 185 190Leu Phe Asp Lys Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala 195 200 205Arg Asp Trp Pro Asp Ala Arg Gly Ile Trp His Asn Asp Asn Lys Thr 210 215 220Phe Leu Val Trp Val Asn Glu Glu Asp His Leu Arg Val Ile Ser Met225 230 235 240Gln Lys Gly Gly Asn Met Lys Glu Val Phe Thr Arg Phe Cys Thr Gly 245 250 255Leu Thr Gln Ile Glu Thr Leu Phe Lys Ser Lys Asp Tyr Glu Phe Met 260 265 270Trp Asn Pro His Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu Gly 275 280 285Thr Gly Leu Arg Ala Gly Val His Ile Lys Leu Pro Asn Leu Gly Lys 290 295 300His Glu Lys Phe Ser Glu Val Leu Lys Arg Leu Arg Leu Gln Lys Arg305 310 315 320Gly Thr Gly Gly Val Asp Thr Ala Ala Val Gly Gly Val Phe Asp Val 325 330 335Ser Asn Ala Asp Arg Leu Gly Phe Ser Glu Val Glu Leu Val Gln Met 340 345 350Val Val Asp Gly Val Lys Leu Leu Ile Glu Met Glu Gln Arg Leu Glu 355 360 365Gln Gly Gln Ala Ile Asp Asp Leu Met Pro Ala Gln Lys 370 375 38021381PRTHomo sapiens 21Met Pro Phe Gly Asn Thr His Asn Lys Phe Lys Leu Asn Tyr Lys Pro1 5 10 15Glu Glu Glu Tyr Pro Asp Leu Ser Lys His Asn Asn His Met Ala Lys 20 25 30Val Leu Thr Leu Glu Leu Tyr Lys Lys Leu Arg Asp Lys Glu Thr Pro 35 40 45Ser Gly Phe Thr Val Asp Asp Val Ile Gln Thr Gly Val Asp Asn Pro 50 55 60Gly His Pro Phe Ile Met Thr Val Gly Cys Val Ala Gly Asp Glu Glu65 70 75 80Ser Tyr Glu Val Phe Lys Glu Leu Phe Asp Pro Ile Ile Ser Asp Arg 85 90 95His Gly Gly Tyr Lys Pro Thr Asp Lys His Lys Thr Asp Leu Asn His 100 105 110Glu Asn Leu Lys Gly Gly Asp Asp Leu Asp Pro Asn Tyr Val Leu Ser 115 120 125Ser Arg Val Arg Thr Gly Arg Ser Ile Lys Gly Tyr Thr Leu Pro Pro 130 135 140His Cys Ser Arg Gly Glu Arg Arg Ala Val Glu Lys Leu Ser Val Glu145 150 155 160Ala Leu Asn Ser Leu Thr Gly Glu Phe Lys Gly Lys Tyr Tyr Pro Leu 165 170 175Lys Ser Met Thr Glu Lys Glu Gln Gln Gln Leu Ile Asp Asp His Phe 180 185 190Leu Phe Asp Lys Pro Val Ser Pro Leu Leu Leu Ala Ser Gly Met Ala 195 200 205Arg Asp Trp Pro Asp Ala Arg Gly Ile Trp His Asn Asp Asn Lys Ser 210 215 220Phe Leu Val Trp Val Asn Glu Glu Asp His Leu Arg Val Ile Ser Met225 230 235 240Glu Lys Gly Gly Asn Met Lys Glu Val Phe Arg Arg Phe Cys Val Gly 245 250 255Leu Gln Lys Ile Glu Glu Ile Phe Lys Lys Ala Gly His Pro Phe Met 260 265 270Trp Asn Gln His Leu Gly Tyr Val Leu Thr Cys Pro Ser Asn Leu Gly 275 280 285Thr Gly Leu Arg Gly Gly Val His Val Lys Leu Ala His Leu Ser Lys 290 295 300His Pro Lys Phe Glu Glu Ile Leu Thr Arg Leu Arg Leu Gln Lys Arg305 310 315 320Gly Thr Gly Gly Val Asp Thr Ala Ala Val Gly Ser Val Phe Asp Val 325 330 335Ser Asn Ala Asp Arg Leu Gly Ser Ser Glu Val Glu Gln Val Gln Leu 340 345 350Val Val Asp Gly Val Lys Leu Met Val Glu Met Glu Lys Lys Leu Glu 355 360 365Lys Gly Gln Ser Ile Asp Asp Met Ile Pro Ala Gln Lys 370 375 38022417PRTHomo sapiens 22Met Ala Gly Pro Phe Ser Arg Leu Leu Ser Ala Arg Pro Gly Leu Arg1 5 10 15Leu Leu Ala Leu Ala Gly Ala Gly Ser Leu Ala Ala Gly Phe Leu Leu 20 25 30Arg Pro Glu Pro Val Arg Ala Ala Ser Glu Arg Arg Arg Leu Tyr Pro 35 40 45Pro Ser Ala Glu Tyr Pro Asp Leu Arg Lys His Asn Asn Cys Met Ala 50 55 60Ser His Leu Thr Pro Ala Val Tyr Ala Arg Leu Cys Asp Lys Thr Thr65 70 75 80Pro Thr Gly Trp Thr Leu Asp Gln Cys Ile Gln Thr Gly Val Asp Asn 85 90 95Pro Gly His Pro Phe Ile Lys Thr Val Gly Met Val Ala Gly Asp Glu 100 105 110Glu Thr Tyr Glu Val Phe Ala Asp Leu Phe Asp Pro Val Ile Gln Glu 115 120 125Arg His Asn Gly Tyr Asp Pro Arg Thr Met Lys His Thr Thr Asp Leu 130 135 140Asp Ala Ser Lys Ile Arg Ser Gly Tyr Phe Asp Glu Arg Tyr Val Leu145 150 155 160Ser Ser Arg Val Arg Thr Gly Arg Ser Ile Arg Gly Leu Ser Leu Pro 165 170 175Pro Ala Cys Thr Arg Ala Glu Arg Arg Glu Val Glu Arg Val Val Val 180 185 190Asp Ala Leu Ser Gly Leu Lys Gly Asp Leu Ala Gly Arg Tyr Tyr Arg 195 200 205Leu Ser Glu Met Thr Glu Ala Glu Gln Gln Gln Leu Ile Asp Asp His 210 215 220Phe Leu Phe Asp Lys Pro Val Ser Pro Leu Leu Thr Ala Ala Gly Met225 230 235 240Ala Arg Asp Trp Pro Asp Ala Arg Gly Ile Trp His Asn Asn Glu Lys 245 250 255Ser Phe Leu Ile Trp Val Asn Glu Glu Asp His Thr Arg Val Ile Ser 260 265 270Met Glu Lys Gly Gly Asn Met Lys Arg Val Phe Glu Arg Phe Cys Arg 275 280 285Gly Leu Lys Glu Val Glu Arg Leu Ile Gln Glu Arg Gly Trp Glu Phe 290 295 300Met Trp Asn Glu Arg Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn Leu305 310 315 320Gly Thr Gly Leu Arg Ala Gly Val His Ile Lys Leu Pro Leu Leu Ser 325 330 335Lys Asp Ser Arg Phe Pro Lys Ile Leu Glu Asn Leu Arg Leu Gln Lys 340 345 350Arg Gly Thr Gly Gly Val Asp Thr Ala Ala Thr Gly Gly Val Phe Asp 355 360 365Ile Ser Asn Leu Asp Arg Leu Gly Lys Ser Glu Val Glu Leu Val Gln 370 375 380Leu Val Ile Asp Gly Val Asn Tyr Leu Ile Asp Cys Glu Arg Arg Leu385 390 395 400Glu Arg Gly Gln Asp Ile Arg Ile Pro Thr Pro Val Ile His Thr Lys 405 410 415His23419PRTHomo sapiens 23Met Ala Ser Ile Phe Ser Lys Leu Leu Thr Gly Arg Asn Ala Ser Leu1 5 10 15Leu Phe Ala Thr Met Gly Thr Ser Val Leu Thr Thr Gly Tyr Leu Leu 20 25 30Asn Arg Gln Lys Val Cys Ala Glu Val Arg Glu Gln Pro Arg Leu Phe 35 40 45Pro Pro Ser Ala Asp Tyr Pro Asp Leu Arg Lys His Asn Asn Cys Met 50 55 60Ala Glu Cys Leu Thr Pro Ala Ile Tyr Ala Lys Leu Arg Asn Lys Val65 70 75 80Thr Pro Asn Gly Tyr Thr Leu Asp Gln Cys Ile Gln Thr Gly Val Asp 85 90 95Asn Pro Gly His Pro Phe Ile Lys Thr Val Gly Met Val Ala Gly Asp 100 105 110Glu Glu Ser Tyr Glu Val Phe Ala Asp Leu Phe Asp Pro Val Ile Lys 115 120 125Leu Arg His Asn Gly Tyr Asp Pro Arg Val Met Lys His Thr Thr Asp 130 135 140Leu Asp Ala Ser Lys Ile Thr Gln Gly Gln Phe Asp Glu His Tyr Val145 150 155 160Leu Ser Ser Arg Val Arg Thr Gly Arg Ser Ile Arg Gly Leu Ser Leu 165 170 175Pro Pro Ala Cys Thr Arg Ala Glu Arg Arg Glu Val Glu Asn Val Ala 180 185 190Ile Thr Ala Leu Glu Gly Leu Lys Gly Asp Leu Ala Gly Arg Tyr Tyr 195 200 205Lys Leu Ser Glu Met Thr Glu Gln Asp Gln Gln Arg Leu Ile Asp Asp 210 215 220His Phe Leu Phe Asp Lys Pro Val Ser Pro Leu Leu Thr Cys Ala Gly225 230 235 240Met Ala Arg Asp Trp Pro Asp Ala Arg Gly Ile Trp His Asn Tyr Asp 245 250 255Lys Thr Phe Leu Ile Trp Ile Asn Glu Glu Asp His Thr Arg Val Ile 260 265 270Ser Met Glu Lys Gly Gly Asn Met Lys Arg Val Phe Glu Arg Phe Cys 275 280 285Arg Gly Leu Lys Glu Val Glu Arg Leu Ile Gln Glu Arg Gly Trp Glu 290 295 300Phe Met Trp Asn Glu Arg Leu Gly Tyr Ile Leu Thr Cys Pro Ser Asn305 310 315 320Leu Gly Thr Gly Leu Arg Ala Gly Val His Val Arg Ile Pro Lys Leu 325 330 335Ser Lys Asp Pro Arg Phe Ser Lys Ile Leu Glu Asn Leu Arg Leu Gln 340 345 350Lys Arg Gly Thr Gly Gly Val Asp Thr Ala Ala Val Ala Asp Val Tyr 355 360 365Asp Ile Ser Asn Ile Asp Arg Ile Gly Arg Ser Glu Val Glu Leu Val 370 375 380Gln Ile Val Ile Asp Gly Val Asn Tyr Leu Val Asp Cys Glu Lys Lys385 390 395 400Leu Glu Arg Gly Gln Asp Ile Lys Val Pro Pro Pro Leu Pro Gln Phe 405 410 415Gly Lys Lys24523PRTMus musculus 24Met Ala Ala Ala Leu Gln Val Leu Pro Cys Leu Leu Arg Ala Pro Ser1 5 10 15Arg Pro Leu Leu Trp Gly Pro Pro Val Ala Arg Met Thr Ser Gly Met 20 25 30Ala Leu Ala Glu Gln Ala Arg Gln Leu Phe Asp Ser Ala Val Gly Ala 35 40 45Val Gln Pro Gly Pro Met Leu Gln Arg Thr Leu Ser Leu Asp Pro Ser 50 55 60Gly Arg Gln Leu Lys Val Arg Asp Arg Thr Phe Gln Leu Arg Glu Asn65 70 75 80Leu Tyr Leu Val Gly Phe Gly Lys Ala Val Leu Gly Met Ala Ala Ala 85 90 95Ala Glu Glu Leu Leu Ala Gln His Leu Val Gln Gly Val Ile Ser Val 100 105 110Pro Lys Gly Ile Arg Ala Ala Met Glu His Ala Gly Lys Lys Glu Met 115 120 125Leu Leu Lys Pro His Ser Arg Val Gln Val Phe Glu Gly Ala Glu Asp 130 135 140Asn Leu Pro Asp Arg Asp Ala Leu Arg Ala Ala Leu Thr Ile Gln Gln145 150 155 160Leu Ala Glu Gly Leu Thr Ala Asp Asp Leu Leu Leu Val Leu Ile Ser 165 170 175Gly Gly Gly Ser Ala Leu Leu Pro Ala Pro Ile Pro Pro Val Thr Leu 180 185 190Glu Glu Lys Gln Met Leu Thr Lys Leu Leu Ala Ala Arg Gly Ala Thr 195 200 205Ile Gln Glu Leu Asn Thr Ile Arg Lys Ala Leu Ser Gln Leu Lys Gly 210 215 220Gly Gly Leu Ala Gln Ala Ala Tyr Pro Ala Gln Val Ile Ser Leu Ile225 230 235 240Leu Ser Asp Val Ile Gly Asp Pro Leu Glu Val Ile Ala Ser Gly Pro 245 250 255Thr Val Ala Ser Ala His Ser Val Gln Asp Cys Leu His Ile Leu Asn 260 265 270His Tyr Gly Leu Arg Ala Ala Leu Pro Arg Ser Val Lys Thr Val Leu 275 280 285Ser Arg Ala Asp Ser Asp Pro His Gly Pro His Thr Cys Gly His Val 290 295 300Leu Asn Val Ile Ile Gly Ser Asn Ser Leu Ala Leu Ala Glu Ala Gln305 310 315 320Arg Gln Ala Glu Val Leu Gly Tyr His Ala Met Val Leu Ser Thr Ala 325 330 335Met Gln Gly Asp Val Lys Arg Val Ala Arg Phe Tyr Gly Leu Leu Ala 340 345 350Arg Val Ala Ala Ala His Leu Thr Pro Ser Leu Ala Glu Arg Pro Leu 355 360 365Glu Glu Glu Ala Glu Leu His Gln Leu Ala Ala Glu Leu Gln Leu Pro 370 375 380Asp Leu Gln Leu Glu Glu Ala Leu Glu Ala Val Ala Lys Ala Lys Gly385 390 395 400Pro Val Cys Leu Leu Ala Gly Gly Glu Pro Thr Val Gln Leu Gln Gly 405 410 415Ser Gly Lys Gly Gly Arg Asn Gln Glu Leu Ala Leu His Val Gly Val 420 425 430Glu Leu Gly Arg Gln Pro Leu Gly Pro Ile Asp Val Leu Phe Leu Ser 435 440 445Gly Gly Thr Asp Gly Gln Asp Gly Pro Thr Lys Val Ala Gly Ala Trp 450 455 460Val Met Ser Asp Leu Ile Ser Gln Ala Ser Ala Glu Ser Leu Asp Ile465 470 475 480Ala Thr Ser Leu Thr Asn Asn Asp Ser Tyr Thr Phe Phe Cys Arg Phe 485 490 495Arg Gly Gly Thr His Leu Leu His Thr Gly Leu Thr Gly Thr Asn Val 500 505 510Met Asp Val His Leu Leu Ile Leu His Pro Gln 515 52025396PRTMus musculus 25Met Leu Leu Lys Pro His Ser Arg Val Gln Val Phe Glu Gly Ala Glu1 5 10 15Asp Asn Leu Pro Asp Arg Asp Ala Leu Arg Ala Ala Leu Thr Ile Gln 20 25 30Gln Leu Ala Glu Gly Leu Thr Ala Asp Asp Leu Leu Leu Val Leu Ile 35 40 45Ser Gly Gly Gly Ser Ala Leu Leu Pro Ala Pro Ile Pro Pro Val Thr 50 55 60Leu Glu Glu Lys Gln Met Leu Thr Lys Leu Leu Ala Ala Arg Gly Ala65 70 75 80Thr Ile Gln Glu Leu Asn Thr Ile Arg Lys Ala Leu Ser Gln Leu Lys 85 90 95Gly Gly Gly Leu Ala Gln Ala Ala Tyr Pro Ala Gln Val Ile Ser Leu 100 105 110Ile Leu Ser Asp Val Ile Gly Asp Pro Leu Glu Val Ile Ala Ser Gly 115 120 125Pro Thr Val Ala Ser Ala His Ser Val Gln Asp Cys Leu His Ile Leu 130 135 140Asn His Tyr Gly Leu Arg Ala Ala Leu Pro Arg Ser Val Lys Thr Val145 150 155 160Leu Ser Arg Ala Asp Ser Asp Pro His Gly Pro His Thr Cys Gly His 165 170 175Val Leu Asn Val Ile Ile Gly Ser Asn Ser Leu Ala Leu Ala Glu Ala 180 185 190Gln Arg Gln Ala Glu Val Leu Gly Tyr His Ala Met Val Leu Ser Thr 195 200 205Ala Met Gln Gly Asp Val Lys Arg Val Ala Arg Phe Tyr Gly Leu Leu 210 215 220Ala Arg Val Ala Ala Ala His Leu Thr Pro Ser Leu Ala Glu Arg Pro225 230 235 240Leu Glu Glu Glu Ala Glu Leu His Gln Leu

Ala Ala Glu Leu Gln Leu 245 250 255Pro Asp Leu Gln Leu Glu Glu Ala Leu Glu Ala Val Ala Lys Ala Lys 260 265 270Gly Pro Val Cys Leu Leu Ala Gly Gly Glu Pro Thr Val Gln Leu Gln 275 280 285Gly Ser Gly Lys Gly Gly Arg Asn Gln Glu Leu Ala Leu His Val Gly 290 295 300Val Glu Leu Gly Arg Gln Pro Leu Gly Pro Ile Asp Val Leu Phe Leu305 310 315 320Ser Gly Gly Thr Asp Gly Gln Asp Gly Pro Thr Lys Val Ala Gly Ala 325 330 335Trp Val Met Ser Asp Leu Ile Ser Gln Ala Ser Ala Glu Ser Leu Asp 340 345 350Ile Ala Thr Ser Leu Thr Asn Asn Asp Ser Tyr Thr Phe Phe Cys Arg 355 360 365Phe Arg Gly Gly Thr His Leu Leu His Thr Gly Leu Thr Gly Thr Asn 370 375 380Val Met Asp Val His Leu Leu Ile Leu His Pro Gln385 390 39526917PRTHomo sapiens 26Met Ile Ala Ala Gln Leu Leu Ala Tyr Tyr Phe Thr Glu Leu Lys Asp1 5 10 15Asp Gln Val Lys Lys Ile Asp Lys Tyr Leu Tyr Ala Met Arg Leu Ser 20 25 30Asp Glu Thr Leu Ile Asp Ile Met Thr Arg Phe Arg Lys Glu Met Lys 35 40 45Asn Gly Leu Ser Arg Asp Phe Asn Pro Thr Ala Thr Val Lys Met Leu 50 55 60Pro Thr Phe Val Arg Ser Ile Pro Asp Gly Ser Glu Lys Gly Asp Phe65 70 75 80Ile Ala Leu Asp Leu Gly Gly Ser Ser Phe Arg Ile Leu Arg Val Gln 85 90 95Val Asn His Glu Lys Asn Gln Asn Val His Met Glu Ser Glu Val Tyr 100 105 110Asp Thr Pro Glu Asn Ile Val His Gly Ser Gly Ser Gln Leu Phe Asp 115 120 125His Val Ala Glu Cys Leu Gly Asp Phe Met Glu Lys Arg Lys Ile Lys 130 135 140Asp Lys Lys Leu Pro Val Gly Phe Thr Phe Ser Phe Pro Cys Gln Gln145 150 155 160Ser Lys Ile Asp Glu Ala Ile Leu Ile Thr Trp Thr Lys Arg Phe Lys 165 170 175Ala Ser Gly Val Glu Gly Ala Asp Val Val Lys Leu Leu Asn Lys Ala 180 185 190Ile Lys Lys Arg Gly Asp Tyr Asp Ala Asn Ile Val Ala Val Val Asn 195 200 205Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr Asp Asp Gln His Cys 210 215 220Glu Val Gly Leu Ile Ile Gly Thr Gly Thr Asn Ala Cys Tyr Met Glu225 230 235 240Glu Leu Arg His Ile Asp Leu Val Glu Gly Asp Glu Gly Arg Met Cys 245 250 255Ile Asn Thr Glu Trp Gly Ala Phe Gly Asp Asp Gly Ser Leu Glu Asp 260 265 270Ile Arg Thr Glu Phe Asp Arg Glu Ile Asp Arg Gly Ser Leu Asn Pro 275 280 285Gly Lys Gln Leu Phe Glu Lys Met Val Ser Gly Met Tyr Leu Gly Glu 290 295 300Leu Val Arg Leu Ile Leu Val Lys Met Ala Lys Glu Gly Leu Leu Phe305 310 315 320Glu Gly Arg Ile Thr Pro Glu Leu Leu Thr Arg Gly Lys Phe Asn Thr 325 330 335Ser Asp Val Ser Ala Ile Glu Lys Asn Lys Glu Gly Leu His Asn Ala 340 345 350Lys Glu Ile Leu Thr Arg Leu Gly Val Glu Pro Ser Asp Asp Asp Cys 355 360 365Val Ser Val Gln His Val Cys Thr Ile Val Ser Phe Arg Ser Ala Asn 370 375 380Leu Val Ala Ala Thr Leu Gly Ala Ile Leu Asn Arg Leu Arg Asp Asn385 390 395 400Lys Gly Thr Pro Arg Leu Arg Thr Thr Val Gly Val Asp Gly Ser Leu 405 410 415Tyr Lys Thr His Pro Gln Tyr Ser Arg Arg Phe His Lys Thr Leu Arg 420 425 430Arg Leu Val Pro Asp Ser Asp Val Arg Phe Leu Leu Ser Glu Ser Gly 435 440 445Ser Gly Lys Gly Ala Ala Met Val Thr Ala Val Ala Tyr Arg Leu Ala 450 455 460Glu Gln His Arg Gln Ile Glu Glu Thr Leu Ala His Phe His Leu Thr465 470 475 480Lys Asp Met Leu Leu Glu Val Lys Lys Arg Met Arg Ala Glu Met Glu 485 490 495Leu Gly Leu Arg Lys Gln Thr His Asn Asn Ala Val Val Lys Met Leu 500 505 510Pro Ser Phe Val Arg Arg Thr Pro Asp Gly Thr Glu Asn Gly Asp Phe 515 520 525Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val Lys 530 535 540Ile Arg Ser Gly Lys Lys Arg Thr Val Glu Met His Asn Lys Ile Tyr545 550 555 560Ala Ile Pro Ile Glu Ile Met Gln Gly Thr Gly Glu Glu Leu Phe Asp 565 570 575His Ile Val Ser Cys Ile Ser Asp Phe Leu Asp Tyr Met Gly Ile Lys 580 585 590Gly Pro Arg Met Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys Gln Gln 595 600 605Thr Ser Leu Asp Ala Gly Ile Leu Ile Thr Trp Thr Lys Gly Phe Lys 610 615 620Ala Thr Asp Cys Val Gly His Asp Val Val Thr Leu Leu Arg Asp Ala625 630 635 640Ile Lys Arg Arg Glu Glu Phe Asp Leu Asp Val Val Ala Val Val Asn 645 650 655Asp Thr Val Gly Thr Met Met Thr Cys Ala Tyr Glu Glu Pro Thr Cys 660 665 670Glu Val Gly Leu Ile Val Gly Thr Gly Ser Asn Ala Cys Tyr Met Glu 675 680 685Glu Met Lys Asn Val Glu Met Val Glu Gly Asp Gln Gly Gln Met Cys 690 695 700Ile Asn Met Glu Trp Gly Ala Phe Gly Asp Asn Gly Cys Leu Asp Asp705 710 715 720Ile Arg Thr His Tyr Asp Arg Leu Val Asp Glu Tyr Ser Leu Asn Ala 725 730 735Gly Lys Gln Arg Tyr Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu 740 745 750Ile Val Arg Asn Ile Leu Ile Asp Phe Thr Lys Lys Gly Phe Leu Phe 755 760 765Arg Gly Gln Ile Ser Glu Thr Leu Lys Thr Arg Gly Ile Phe Glu Thr 770 775 780Lys Phe Leu Ser Gln Ile Glu Ser Asp Arg Leu Ala Leu Leu Gln Val785 790 795 800Arg Ala Ile Leu Gln Gln Leu Gly Leu Asn Ser Thr Cys Asp Asp Ser 805 810 815Ile Leu Val Lys Thr Val Cys Gly Val Val Ser Arg Arg Ala Ala Gln 820 825 830Leu Cys Gly Ala Gly Met Ala Ala Val Val Asp Lys Ile Arg Glu Asn 835 840 845Arg Gly Leu Asp Arg Leu Asn Val Thr Val Gly Val Asp Gly Thr Leu 850 855 860Tyr Lys Leu His Pro His Phe Ser Arg Ile Met His Gln Thr Val Lys865 870 875 880Glu Leu Ser Pro Lys Cys Asn Val Ser Phe Leu Leu Ser Glu Asp Gly 885 890 895Ser Gly Lys Gly Ala Ala Leu Ile Thr Ala Val Gly Val Arg Leu Arg 900 905 910Thr Glu Ala Ser Ser 91527917PRTHomo sapiens 27Met Ile Ala Ser His Leu Leu Ala Tyr Phe Phe Thr Glu Leu Asn His1 5 10 15Asp Gln Val Gln Lys Val Asp Gln Tyr Leu Tyr His Met Arg Leu Ser 20 25 30Asp Glu Thr Leu Leu Glu Ile Ser Lys Arg Phe Arg Lys Glu Met Glu 35 40 45Lys Gly Leu Gly Ala Thr Thr His Pro Thr Ala Ala Val Lys Met Leu 50 55 60Pro Thr Phe Val Arg Ser Thr Pro Asp Gly Thr Glu His Gly Glu Phe65 70 75 80Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Trp Val Lys 85 90 95Val Thr Asp Asn Gly Leu Gln Lys Val Glu Met Glu Asn Gln Ile Tyr 100 105 110Ala Ile Pro Glu Asp Ile Met Arg Gly Ser Gly Thr Gln Leu Phe Asp 115 120 125His Ile Ala Glu Cys Leu Ala Asn Phe Met Asp Lys Leu Gln Ile Lys 130 135 140Asp Lys Lys Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys His Gln145 150 155 160Thr Lys Leu Asp Glu Ser Phe Leu Val Ser Trp Thr Lys Gly Phe Lys 165 170 175Ser Ser Gly Val Glu Gly Arg Asp Val Val Ala Leu Ile Arg Lys Ala 180 185 190Ile Gln Arg Arg Gly Asp Phe Asp Ile Asp Ile Val Ala Val Val Asn 195 200 205Asp Thr Val Gly Thr Met Met Thr Cys Gly Tyr Asp Asp His Asn Cys 210 215 220Glu Ile Gly Leu Ile Val Gly Thr Gly Ser Asn Ala Cys Tyr Met Glu225 230 235 240Glu Met Arg His Ile Asp Met Val Glu Gly Asp Glu Gly Arg Met Cys 245 250 255Ile Asn Met Glu Trp Gly Ala Phe Gly Asp Asp Gly Ser Leu Asn Asp 260 265 270Ile Arg Thr Glu Phe Asp Gln Glu Ile Asp Met Gly Ser Leu Asn Pro 275 280 285Gly Lys Gln Leu Phe Glu Lys Met Ile Ser Gly Met Tyr Met Gly Glu 290 295 300Leu Val Arg Leu Ile Leu Val Lys Met Ala Lys Glu Glu Leu Leu Phe305 310 315 320Gly Gly Lys Leu Ser Pro Glu Leu Leu Asn Thr Gly Arg Phe Glu Thr 325 330 335Lys Asp Ile Ser Asp Ile Glu Gly Glu Lys Asp Gly Ile Arg Lys Ala 340 345 350Arg Glu Val Leu Met Arg Leu Gly Leu Asp Pro Thr Gln Glu Asp Cys 355 360 365Val Ala Thr His Arg Ile Cys Gln Ile Val Ser Thr Arg Ser Ala Ser 370 375 380Leu Cys Ala Ala Thr Leu Ala Ala Val Leu Gln Arg Ile Lys Glu Asn385 390 395 400Lys Gly Glu Glu Arg Leu Arg Ser Thr Ile Gly Val Asp Gly Ser Val 405 410 415Tyr Lys Lys His Pro His Phe Ala Lys Arg Leu His Lys Thr Val Arg 420 425 430Arg Leu Val Pro Gly Cys Asp Val Arg Phe Leu Arg Ser Glu Asp Gly 435 440 445Ser Gly Lys Gly Ala Ala Met Val Thr Ala Val Ala Tyr Arg Leu Ala 450 455 460Asp Gln His Arg Ala Arg Gln Lys Thr Leu Glu His Leu Gln Leu Ser465 470 475 480His Asp Gln Leu Leu Glu Val Lys Arg Arg Met Lys Val Glu Met Glu 485 490 495Arg Gly Leu Ser Lys Glu Thr His Ala Ser Ala Pro Val Lys Met Leu 500 505 510Pro Thr Tyr Val Cys Ala Thr Pro Asp Gly Thr Glu Lys Gly Asp Phe 515 520 525Leu Ala Leu Asp Leu Gly Gly Thr Asn Phe Arg Val Leu Leu Val Arg 530 535 540Val Arg Asn Gly Lys Trp Gly Gly Val Glu Met His Asn Lys Ile Tyr545 550 555 560Ala Ile Pro Gln Glu Val Met His Gly Thr Gly Asp Glu Leu Phe Asp 565 570 575His Ile Val Gln Cys Ile Ala Asp Phe Leu Glu Tyr Met Gly Met Lys 580 585 590Gly Val Ser Leu Pro Leu Gly Phe Thr Phe Ser Phe Pro Cys Gln Gln 595 600 605Asn Ser Leu Asp Glu Ser Ile Leu Leu Lys Trp Thr Lys Gly Phe Lys 610 615 620Ala Ser Gly Cys Glu Gly Glu Asp Val Val Thr Leu Leu Lys Glu Ala625 630 635 640Ile His Arg Arg Glu Glu Phe Asp Leu Asp Val Val Ala Val Val Asn 645 650 655Asp Thr Val Gly Thr Met Met Thr Cys Gly Phe Glu Asp Pro His Cys 660 665 670Glu Val Gly Leu Ile Val Gly Thr Gly Ser Asn Ala Cys Tyr Met Glu 675 680 685Glu Met Arg Asn Val Glu Leu Val Glu Gly Glu Glu Gly Arg Met Cys 690 695 700Val Asn Met Glu Trp Gly Ala Phe Gly Asp Asn Gly Cys Leu Asp Asp705 710 715 720Phe Arg Thr Glu Phe Asp Val Ala Val Asp Glu Leu Ser Leu Asn Pro 725 730 735Gly Lys Gln Arg Phe Glu Lys Met Ile Ser Gly Met Tyr Leu Gly Glu 740 745 750Ile Val Arg Asn Ile Leu Ile Asp Phe Thr Lys Arg Gly Leu Leu Phe 755 760 765Arg Gly Arg Ile Ser Glu Arg Leu Lys Thr Arg Gly Ile Phe Glu Thr 770 775 780Lys Phe Leu Ser Gln Ile Glu Ser Asp Cys Leu Ala Leu Leu Gln Val785 790 795 800Arg Ala Ile Leu Gln His Leu Gly Leu Glu Ser Thr Cys Asp Asp Ser 805 810 815Ile Ile Val Lys Glu Val Cys Thr Val Val Ala Arg Arg Ala Ala Gln 820 825 830Leu Cys Gly Ala Gly Met Ala Ala Val Val Asp Arg Ile Arg Glu Asn 835 840 845Arg Gly Leu Asp Ala Leu Lys Val Thr Val Gly Val Asp Gly Thr Leu 850 855 860Tyr Lys Leu His Pro His Phe Ala Lys Val Met His Glu Thr Val Lys865 870 875 880Asp Leu Ala Pro Lys Cys Asp Val Ser Phe Leu Gln Ser Glu Asp Gly 885 890 895Ser Gly Lys Gly Ala Ala Leu Ile Thr Ala Val Ala Cys Arg Ile Arg 900 905 910Glu Ala Gly Gln Arg 91528923PRTHomo sapiens 28Met Asp Ser Ile Gly Ser Ser Gly Leu Arg Gln Gly Glu Glu Thr Leu1 5 10 15Ser Cys Ser Glu Glu Gly Leu Pro Gly Pro Ser Asp Ser Ser Glu Leu 20 25 30Val Gln Glu Cys Leu Gln Gln Phe Lys Val Thr Arg Ala Gln Leu Gln 35 40 45Gln Ile Gln Ala Ser Leu Leu Gly Ser Met Glu Gln Ala Leu Arg Gly 50 55 60Gln Ala Ser Pro Ala Pro Ala Val Arg Met Leu Pro Thr Tyr Val Gly65 70 75 80Ser Thr Pro His Gly Thr Glu Gln Gly Asp Phe Val Val Leu Glu Leu 85 90 95Gly Ala Thr Gly Ala Ser Leu Arg Val Leu Trp Val Thr Leu Thr Gly 100 105 110Ile Glu Gly His Arg Val Glu Pro Arg Ser Gln Glu Phe Val Ile Pro 115 120 125Gln Glu Val Met Leu Gly Ala Gly Gln Gln Leu Phe Asp Phe Ala Ala 130 135 140His Cys Leu Ser Glu Phe Leu Asp Ala Gln Pro Val Asn Lys Gln Gly145 150 155 160Leu Gln Leu Gly Phe Ser Phe Ser Phe Pro Cys His Gln Thr Gly Leu 165 170 175Asp Arg Ser Thr Leu Ile Ser Trp Thr Lys Gly Phe Arg Cys Ser Gly 180 185 190Val Glu Gly Gln Asp Val Val Gln Leu Leu Arg Asp Ala Ile Arg Arg 195 200 205Gln Gly Ala Tyr Asn Ile Asp Val Val Ala Val Val Asn Asp Thr Val 210 215 220Gly Thr Met Met Gly Cys Glu Pro Gly Val Arg Pro Cys Glu Val Gly225 230 235 240Leu Val Val Asp Thr Gly Thr Asn Ala Cys Tyr Met Glu Glu Ala Arg 245 250 255His Val Ala Val Leu Asp Glu Asp Arg Gly Arg Val Cys Val Ser Val 260 265 270Glu Trp Gly Ser Phe Ser Asp Asp Gly Ala Leu Gly Pro Val Leu Thr 275 280 285Thr Phe Asp His Thr Leu Asp His Glu Ser Leu Asn Pro Gly Ala Gln 290 295 300Arg Phe Glu Lys Met Ile Gly Gly Leu Tyr Leu Gly Glu Leu Val Arg305 310 315 320Leu Val Leu Ala His Leu Ala Arg Cys Gly Val Leu Phe Gly Gly Cys 325 330 335Thr Ser Pro Ala Leu Leu Ser Gln Gly Ser Ile Leu Leu Glu His Val 340 345 350Ala Glu Met Glu Asp Pro Ser Thr Gly Ala Ala Arg Val His Ala Ile 355 360 365Leu Gln Asp Leu Gly Leu Ser Pro Gly Ala Ser Asp Val Glu Leu Val 370 375 380Gln His Val Cys Ala Ala Val Cys Thr Arg Ala Ala Gln Leu Cys Ala385 390 395 400Ala Ala Leu Ala Ala Val Leu Ser Cys Leu Gln His Ser Arg Glu Gln 405 410 415Gln Thr Leu Gln Val Ala Val Ala Thr Gly Gly Arg Val Cys Glu Arg 420 425 430His Pro Arg Phe Cys Ser Val Leu Gln Gly Thr Val Met Leu Leu Ala 435 440 445Pro Glu Cys Asp Val Ser Leu Ile Pro Ser Val Asp Gly Gly Gly Arg 450 455 460Gly Val Ala Met Val Thr Ala Val Ala Ala Arg Leu Ala Ala His Arg465 470 475 480Arg Leu

Leu Glu Glu Thr Leu Ala Pro Phe Arg Leu Asn His Asp Gln 485 490 495Leu Ala Ala Val Gln Ala Gln Met Arg Lys Ala Met Ala Lys Gly Leu 500 505 510Arg Gly Glu Ala Ser Ser Leu Arg Met Leu Pro Thr Phe Val Arg Ala 515 520 525Thr Pro Asp Gly Ser Glu Arg Gly Asp Phe Leu Ala Leu Asp Leu Gly 530 535 540Gly Thr Asn Phe Arg Val Leu Leu Val Arg Val Thr Thr Gly Val Gln545 550 555 560Ile Thr Ser Glu Ile Tyr Ser Ile Pro Glu Thr Val Ala Gln Gly Ser 565 570 575Gly Gln Gln Leu Phe Asp His Ile Val Asp Cys Ile Val Asp Phe Gln 580 585 590Gln Lys Gln Gly Leu Ser Gly Gln Ser Leu Pro Leu Gly Phe Thr Phe 595 600 605Ser Phe Pro Cys Arg Gln Leu Gly Leu Asp Gln Gly Ile Leu Leu Asn 610 615 620Trp Thr Lys Gly Phe Lys Ala Ser Asp Cys Glu Gly Gln Asp Val Val625 630 635 640Ser Leu Leu Arg Glu Ala Ile Thr Arg Arg Gln Ala Val Glu Leu Asn 645 650 655Val Val Ala Ile Val Asn Asp Thr Val Gly Thr Met Met Ser Cys Gly 660 665 670Tyr Glu Asp Pro Arg Cys Glu Ile Gly Leu Ile Val Gly Thr Gly Thr 675 680 685Asn Ala Cys Tyr Met Glu Glu Leu Arg Asn Val Ala Gly Val Pro Gly 690 695 700Asp Ser Gly Arg Met Cys Ile Asn Met Glu Trp Gly Ala Phe Gly Asp705 710 715 720Asp Gly Ser Leu Ala Met Leu Ser Thr Arg Phe Asp Ala Ser Val Asp 725 730 735Gln Ala Ser Ile Asn Pro Gly Lys Gln Arg Phe Glu Lys Met Ile Ser 740 745 750Gly Met Tyr Leu Gly Glu Ile Val Arg His Ile Leu Leu His Leu Thr 755 760 765Ser Leu Gly Val Leu Phe Arg Gly Gln Gln Ile Gln Arg Leu Gln Thr 770 775 780Arg Asp Ile Phe Lys Thr Lys Phe Leu Ser Glu Ile Glu Ser Asp Ser785 790 795 800Leu Ala Leu Arg Gln Val Arg Ala Ile Leu Glu Asp Leu Gly Leu Pro 805 810 815Leu Thr Ser Asp Asp Ala Leu Met Val Leu Glu Val Cys Gln Ala Val 820 825 830Ser Gln Arg Ala Ala Gln Leu Cys Gly Ala Gly Val Ala Ala Val Val 835 840 845Glu Lys Ile Arg Glu Asn Arg Gly Leu Glu Glu Leu Ala Val Ser Val 850 855 860Gly Val Asp Gly Thr Leu Tyr Lys Leu His Pro Arg Phe Ser Ser Leu865 870 875 880Val Ala Ala Thr Val Arg Glu Leu Ala Pro Arg Cys Val Val Thr Phe 885 890 895Leu Gln Ser Glu Asp Gly Ser Gly Lys Gly Ala Ala Leu Val Thr Ala 900 905 910Val Ala Cys Arg Leu Ala Gln Leu Thr Arg Val 915 92029465PRTHomo sapiens 29Met Leu Asp Asp Arg Ala Arg Met Glu Ala Ala Lys Lys Glu Lys Val1 5 10 15Glu Gln Ile Leu Ala Glu Phe Gln Leu Gln Glu Glu Asp Leu Lys Lys 20 25 30Val Met Arg Arg Met Gln Lys Glu Met Asp Arg Gly Leu Arg Leu Glu 35 40 45Thr His Glu Glu Ala Ser Val Lys Met Leu Pro Thr Tyr Val Arg Ser 50 55 60Thr Pro Glu Gly Ser Glu Val Gly Asp Phe Leu Ser Leu Asp Leu Gly65 70 75 80Gly Thr Asn Phe Arg Val Met Leu Val Lys Val Gly Glu Gly Glu Glu 85 90 95Gly Gln Trp Ser Val Lys Thr Lys His Gln Met Tyr Ser Ile Pro Glu 100 105 110Asp Ala Met Thr Gly Thr Ala Glu Met Leu Phe Asp Tyr Ile Ser Glu 115 120 125Cys Ile Ser Asp Phe Leu Asp Lys His Gln Met Lys His Lys Lys Leu 130 135 140Pro Leu Gly Phe Thr Phe Ser Phe Pro Val Arg His Glu Asp Ile Asp145 150 155 160Lys Gly Ile Leu Leu Asn Trp Thr Lys Gly Phe Lys Ala Ser Gly Ala 165 170 175Glu Gly Asn Asn Val Val Gly Leu Leu Arg Asp Ala Ile Lys Arg Arg 180 185 190Gly Asp Phe Glu Met Asp Val Val Ala Met Val Asn Asp Thr Val Ala 195 200 205Thr Met Ile Ser Cys Tyr Tyr Glu Asp His Gln Cys Glu Val Gly Met 210 215 220Ile Val Gly Thr Gly Cys Asn Ala Cys Tyr Met Glu Glu Met Gln Asn225 230 235 240Val Glu Leu Val Glu Gly Asp Glu Gly Arg Met Cys Val Asn Thr Glu 245 250 255Trp Gly Ala Phe Gly Asp Ser Gly Glu Leu Asp Glu Phe Leu Leu Glu 260 265 270Tyr Asp Arg Leu Val Asp Glu Ser Ser Ala Asn Pro Gly Gln Gln Leu 275 280 285Tyr Glu Lys Leu Ile Gly Gly Lys Tyr Met Gly Glu Leu Val Arg Leu 290 295 300Val Leu Leu Arg Leu Val Asp Glu Asn Leu Leu Phe His Gly Glu Ala305 310 315 320Ser Glu Gln Leu Arg Thr Arg Gly Ala Phe Glu Thr Arg Phe Val Ser 325 330 335Gln Val Glu Ser Asp Thr Gly Asp Arg Lys Gln Ile Tyr Asn Ile Leu 340 345 350Ser Thr Leu Gly Leu Arg Pro Ser Thr Thr Asp Cys Asp Ile Val Arg 355 360 365Arg Ala Cys Glu Ser Val Ser Thr Arg Ala Ala His Met Cys Ser Ala 370 375 380Gly Leu Ala Gly Val Ile Asn Arg Met Arg Glu Ser Arg Ser Glu Asp385 390 395 400Val Met Arg Ile Thr Val Gly Val Asp Gly Ser Val Tyr Lys Leu His 405 410 415Pro Ser Phe Lys Glu Arg Phe His Ala Ser Val Arg Arg Leu Thr Pro 420 425 430Ser Cys Glu Ile Thr Phe Ile Glu Ser Glu Glu Gly Ser Gly Arg Gly 435 440 445Ala Ala Leu Val Ser Ala Val Ala Cys Lys Lys Ala Cys Met Leu Gly 450 455 460Gln46530820PRTEscherichia coli 30Met Arg Val Leu Lys Phe Gly Gly Thr Ser Val Ala Asn Ala Glu Arg1 5 10 15Phe Leu Arg Val Ala Asp Ile Leu Glu Ser Asn Ala Arg Gln Gly Gln 20 25 30Val Ala Thr Val Leu Ser Ala Pro Ala Lys Ile Thr Asn His Leu Val 35 40 45Ala Met Ile Glu Lys Thr Ile Ser Gly Gln Asp Ala Leu Pro Asn Ile 50 55 60Ser Asp Ala Glu Arg Ile Phe Ala Glu Leu Leu Thr Gly Leu Ala Ala65 70 75 80Ala Gln Pro Gly Phe Pro Leu Ala Gln Leu Lys Thr Phe Val Asp Gln 85 90 95Glu Phe Ala Gln Ile Lys His Val Leu His Gly Ile Ser Leu Leu Gly 100 105 110Gln Cys Pro Asp Ser Ile Asn Ala Ala Leu Ile Cys Arg Gly Glu Lys 115 120 125Met Ser Ile Ala Ile Met Ala Gly Val Leu Glu Ala Arg Gly His Asn 130 135 140Val Thr Val Ile Asp Pro Val Glu Lys Leu Leu Ala Val Gly His Tyr145 150 155 160Leu Glu Ser Thr Val Asp Ile Ala Glu Ser Thr Arg Arg Ile Ala Ala 165 170 175Ser Arg Ile Pro Ala Asp His Met Val Leu Met Ala Gly Phe Thr Ala 180 185 190Gly Asn Glu Lys Gly Glu Leu Val Val Leu Gly Arg Asn Gly Ser Asp 195 200 205Tyr Ser Ala Ala Val Leu Ala Ala Cys Leu Arg Ala Asp Cys Cys Glu 210 215 220Ile Trp Thr Asp Val Asp Gly Val Tyr Thr Cys Asp Pro Arg Gln Val225 230 235 240Pro Asp Ala Arg Leu Leu Lys Ser Met Ser Tyr Gln Glu Ala Met Glu 245 250 255Leu Ser Tyr Phe Gly Ala Lys Val Leu His Pro Arg Thr Ile Thr Pro 260 265 270Ile Ala Gln Phe Gln Ile Pro Cys Leu Ile Lys Asn Thr Gly Asn Pro 275 280 285Gln Ala Pro Gly Thr Leu Ile Gly Ala Ser Arg Asp Glu Asp Glu Leu 290 295 300Pro Val Lys Gly Ile Ser Asn Leu Asn Asn Met Ala Met Phe Ser Val305 310 315 320Ser Gly Pro Gly Met Lys Gly Met Val Gly Met Ala Ala Arg Val Phe 325 330 335Ala Ala Met Ser Arg Ala Arg Ile Ser Val Val Leu Ile Thr Gln Ser 340 345 350Ser Ser Glu Tyr Ser Ile Ser Phe Cys Val Pro Gln Ser Asp Cys Val 355 360 365Arg Ala Glu Arg Ala Met Gln Glu Glu Phe Tyr Leu Glu Leu Lys Glu 370 375 380Gly Leu Leu Glu Pro Leu Ala Val Thr Glu Arg Leu Ala Ile Ile Ser385 390 395 400Val Val Gly Asp Gly Met Arg Thr Leu Arg Gly Ile Ser Ala Lys Phe 405 410 415Phe Ala Ala Leu Ala Arg Ala Asn Ile Asn Ile Val Ala Ile Ala Gln 420 425 430Gly Ser Ser Glu Arg Ser Ile Ser Val Val Val Asn Asn Asp Asp Ala 435 440 445Thr Thr Gly Val Arg Val Thr His Gln Met Leu Phe Asn Thr Asp Gln 450 455 460Val Ile Glu Val Phe Val Ile Gly Val Gly Gly Val Gly Gly Ala Leu465 470 475 480Leu Glu Gln Leu Lys Arg Gln Gln Ser Trp Leu Lys Asn Lys His Ile 485 490 495Asp Leu Arg Val Cys Gly Val Ala Asn Ser Lys Ala Leu Leu Thr Asn 500 505 510Val His Gly Leu Asn Leu Glu Asn Trp Gln Glu Glu Leu Ala Gln Ala 515 520 525Lys Glu Pro Phe Asn Leu Gly Arg Leu Ile Arg Leu Val Lys Glu Tyr 530 535 540His Leu Leu Asn Pro Val Ile Val Asp Cys Thr Ser Ser Gln Ala Val545 550 555 560Ala Asp Gln Tyr Ala Asp Phe Leu Arg Glu Gly Phe His Val Val Thr 565 570 575Pro Asn Lys Lys Ala Asn Thr Ser Ser Met Asp Tyr Tyr His Gln Leu 580 585 590Arg Tyr Ala Ala Glu Lys Ser Arg Arg Lys Phe Leu Tyr Asp Thr Asn 595 600 605Val Gly Ala Gly Leu Pro Val Ile Glu Asn Leu Gln Asn Leu Leu Asn 610 615 620Ala Gly Asp Glu Leu Met Lys Phe Ser Gly Ile Leu Ser Gly Ser Leu625 630 635 640Ser Tyr Ile Phe Gly Lys Leu Asp Glu Gly Met Ser Phe Ser Glu Ala 645 650 655Thr Thr Leu Ala Arg Glu Met Gly Tyr Thr Glu Pro Asp Pro Arg Asp 660 665 670Asp Leu Ser Gly Met Asp Val Ala Arg Lys Leu Leu Ile Leu Ala Arg 675 680 685Glu Thr Gly Arg Glu Leu Glu Leu Ala Asp Ile Glu Ile Glu Pro Val 690 695 700Leu Pro Ala Glu Phe Asn Ala Glu Gly Asp Val Ala Ala Phe Met Ala705 710 715 720Asn Leu Ser Gln Leu Asp Asp Leu Phe Ala Ala Arg Val Ala Lys Ala 725 730 735Arg Asp Glu Gly Lys Val Leu Arg Tyr Val Gly Asn Ile Asp Glu Asp 740 745 750Gly Val Cys Arg Val Lys Ile Ala Glu Val Asp Gly Asn Asp Pro Leu 755 760 765Phe Lys Val Lys Asn Gly Glu Asn Ala Leu Ala Phe Tyr Ser His Tyr 770 775 780Tyr Gln Pro Leu Pro Leu Val Leu Arg Gly Tyr Gly Ala Gly Asn Asp785 790 795 800Val Thr Ala Ala Gly Val Phe Ala Asp Leu Leu Arg Thr Leu Ser Trp 805 810 815Lys Leu Gly Val 82031810PRTEscherichia coli 31Met Ser Val Ile Ala Gln Ala Gly Ala Lys Gly Arg Gln Leu His Lys1 5 10 15Phe Gly Gly Ser Ser Leu Ala Asp Val Lys Cys Tyr Leu Arg Val Ala 20 25 30Gly Ile Met Ala Glu Tyr Ser Gln Pro Asp Asp Met Met Val Val Ser 35 40 45Ala Ala Gly Ser Thr Thr Asn Gln Leu Ile Asn Trp Leu Lys Leu Ser 50 55 60Gln Thr Asp Arg Leu Ser Ala His Gln Val Gln Gln Thr Leu Arg Arg65 70 75 80Tyr Gln Cys Asp Leu Ile Ser Gly Leu Leu Pro Ala Glu Glu Ala Asp 85 90 95Ser Leu Ile Ser Ala Phe Val Ser Asp Leu Glu Arg Leu Ala Ala Leu 100 105 110Leu Asp Ser Gly Ile Asn Asp Ala Val Tyr Ala Glu Val Val Gly His 115 120 125Gly Glu Val Trp Ser Ala Arg Leu Met Ser Ala Val Leu Asn Gln Gln 130 135 140Gly Leu Pro Ala Ala Trp Leu Asp Ala Arg Glu Phe Leu Arg Ala Glu145 150 155 160Arg Ala Ala Gln Pro Gln Val Asp Glu Gly Leu Ser Tyr Pro Leu Leu 165 170 175Gln Gln Leu Leu Val Gln His Pro Gly Lys Arg Leu Val Val Thr Gly 180 185 190Phe Ile Ser Arg Asn Asn Ala Gly Glu Thr Val Leu Leu Gly Arg Asn 195 200 205Gly Ser Asp Tyr Ser Ala Thr Gln Ile Gly Ala Leu Ala Gly Val Ser 210 215 220Arg Val Thr Ile Trp Ser Asp Val Ala Gly Val Tyr Ser Ala Asp Pro225 230 235 240Arg Lys Val Lys Asp Ala Cys Leu Leu Pro Leu Leu Arg Leu Asp Glu 245 250 255Ala Ser Glu Leu Ala Arg Leu Ala Ala Pro Val Leu His Ala Arg Thr 260 265 270Leu Gln Pro Val Ser Gly Ser Glu Ile Asp Leu Gln Leu Arg Cys Ser 275 280 285Tyr Thr Pro Asp Gln Gly Ser Thr Arg Ile Glu Arg Val Leu Ala Ser 290 295 300Gly Thr Gly Ala Arg Ile Val Thr Ser His Asp Asp Val Cys Leu Ile305 310 315 320Glu Phe Gln Val Pro Ala Ser Gln Asp Phe Lys Leu Ala His Lys Glu 325 330 335Ile Asp Gln Ile Leu Lys Arg Ala Gln Val Arg Pro Leu Ala Val Gly 340 345 350Val His Asn Asp Arg Gln Leu Leu Gln Phe Cys Tyr Thr Ser Glu Val 355 360 365Ala Asp Ser Ala Leu Lys Ile Leu Asp Glu Ala Gly Leu Pro Gly Glu 370 375 380Leu Arg Leu Arg Gln Gly Leu Ala Leu Val Ala Met Val Gly Ala Gly385 390 395 400Val Thr Arg Asn Pro Leu His Cys His Arg Phe Trp Gln Gln Leu Lys 405 410 415Gly Gln Pro Val Glu Phe Thr Trp Gln Ser Asp Asp Gly Ile Ser Leu 420 425 430Val Ala Val Leu Arg Thr Gly Pro Thr Glu Ser Leu Ile Gln Gly Leu 435 440 445His Gln Ser Val Phe Arg Ala Glu Lys Arg Ile Gly Leu Val Leu Phe 450 455 460Gly Lys Gly Asn Ile Gly Ser Arg Trp Leu Glu Leu Phe Ala Arg Glu465 470 475 480Gln Ser Thr Leu Ser Ala Arg Thr Gly Phe Glu Phe Val Leu Ala Gly 485 490 495Val Val Asp Ser Arg Arg Ser Leu Leu Ser Tyr Asp Gly Leu Asp Ala 500 505 510Ser Arg Ala Leu Ala Phe Phe Asn Asp Glu Ala Val Glu Gln Asp Glu 515 520 525Glu Ser Leu Phe Leu Trp Met Arg Ala His Pro Tyr Asp Asp Leu Val 530 535 540Val Leu Asp Val Thr Ala Ser Gln Gln Leu Ala Asp Gln Tyr Leu Asp545 550 555 560Phe Ala Ser His Gly Phe His Val Ile Ser Ala Asn Lys Leu Ala Gly 565 570 575Ala Ser Asp Ser Asn Lys Tyr Arg Gln Ile His Asp Ala Phe Glu Lys 580 585 590Thr Gly Arg His Trp Leu Tyr Asn Ala Thr Val Gly Ala Gly Leu Pro 595 600 605Ile Asn His Thr Val Arg Asp Leu Ile Asp Ser Gly Asp Thr Ile Leu 610 615 620Ser Ile Ser Gly Ile Phe Ser Gly Thr Leu Ser Trp Leu Phe Leu Gln625 630 635 640Phe Asp Gly Ser Val Pro Phe Thr Glu Leu Val Asp Gln Ala Trp Gln 645 650 655Gln Gly Leu Thr Glu Pro Asp Pro Arg Asp Asp Leu Ser Gly Lys Asp 660 665 670Val Met Arg Lys Leu Val Ile Leu Ala Arg Glu Ala Gly Tyr Asn Ile 675 680 685Glu Pro Asp Gln Val Arg Val Glu Ser Leu Val Pro Ala His Cys Glu 690 695 700Gly Gly Ser Ile Asp His Phe Phe Glu Asn Gly Asp Glu Leu Asn Glu705 710 715 720Gln Met Val Gln Arg Leu Glu Ala Ala Arg Glu Met Gly Leu Val Leu 725 730 735Arg Tyr Val Ala Arg

Phe Asp Ala Asn Gly Lys Ala Arg Val Gly Val 740 745 750Glu Ala Val Arg Glu Asp His Pro Leu Ala Ser Leu Leu Pro Cys Asp 755 760 765Asn Val Phe Ala Ile Glu Ser Arg Trp Tyr Arg Asp Asn Pro Leu Val 770 775 780Ile Arg Gly Pro Gly Ala Gly Arg Asp Val Thr Ala Gly Ala Ile Gln785 790 795 800Ser Asp Ile Asn Arg Leu Ala Gln Leu Leu 805 81032449PRTEscherichia coli 32Met Ser Glu Ile Val Val Ser Lys Phe Gly Gly Thr Ser Val Ala Asp1 5 10 15Phe Asp Ala Met Asn Arg Ser Ala Asp Ile Val Leu Ser Asp Ala Asn 20 25 30Val Arg Leu Val Val Leu Ser Ala Ser Ala Gly Ile Thr Asn Leu Leu 35 40 45Val Ala Leu Ala Glu Gly Leu Glu Pro Gly Glu Arg Phe Glu Lys Leu 50 55 60Asp Ala Ile Arg Asn Ile Gln Phe Ala Ile Leu Glu Arg Leu Arg Tyr65 70 75 80Pro Asn Val Ile Arg Glu Glu Ile Glu Arg Leu Leu Glu Asn Ile Thr 85 90 95Val Leu Ala Glu Ala Ala Ala Leu Ala Thr Ser Pro Ala Leu Thr Asp 100 105 110Glu Leu Val Ser His Gly Glu Leu Met Ser Thr Leu Leu Phe Val Glu 115 120 125Ile Leu Arg Glu Arg Asp Val Gln Ala Gln Trp Phe Asp Val Arg Lys 130 135 140Val Met Arg Thr Asn Asp Arg Phe Gly Arg Ala Glu Pro Asp Ile Ala145 150 155 160Ala Leu Ala Glu Leu Ala Ala Leu Gln Leu Leu Pro Arg Leu Asn Glu 165 170 175Gly Leu Val Ile Thr Gln Gly Phe Ile Gly Ser Glu Asn Lys Gly Arg 180 185 190Thr Thr Thr Leu Gly Arg Gly Gly Ser Asp Tyr Thr Ala Ala Leu Leu 195 200 205Ala Glu Ala Leu His Ala Ser Arg Val Asp Ile Trp Thr Asp Val Pro 210 215 220Gly Ile Tyr Thr Thr Asp Pro Arg Val Val Ser Ala Ala Lys Arg Ile225 230 235 240Asp Glu Ile Ala Phe Ala Glu Ala Ala Glu Met Ala Thr Phe Gly Ala 245 250 255Lys Val Leu His Pro Ala Thr Leu Leu Pro Ala Val Arg Ser Asp Ile 260 265 270Pro Val Phe Val Gly Ser Ser Lys Asp Pro Arg Ala Gly Gly Thr Leu 275 280 285Val Cys Asn Lys Thr Glu Asn Pro Pro Leu Phe Arg Ala Leu Ala Leu 290 295 300Arg Arg Asn Gln Thr Leu Leu Thr Leu His Ser Leu Asn Met Leu His305 310 315 320Ser Arg Gly Phe Leu Ala Glu Val Phe Gly Ile Leu Ala Arg His Asn 325 330 335Ile Ser Val Asp Leu Ile Thr Thr Ser Glu Val Ser Val Ala Leu Thr 340 345 350Leu Asp Thr Thr Gly Ser Thr Ser Thr Gly Asp Thr Leu Leu Thr Gln 355 360 365Ser Leu Leu Met Glu Leu Ser Ala Leu Cys Arg Val Glu Val Glu Glu 370 375 380Gly Leu Ala Leu Val Ala Leu Ile Gly Asn Asp Leu Ser Lys Ala Cys385 390 395 400Gly Val Gly Lys Glu Val Phe Gly Val Leu Glu Pro Phe Asn Ile Arg 405 410 415Met Ile Cys Tyr Gly Ala Ser Ser His Asn Leu Cys Phe Leu Val Pro 420 425 430Gly Glu Asp Ala Glu Gln Val Val Gln Lys Leu His Ser Asn Leu Phe 435 440 445Glu 33468PRTSalmonella typhimurium 33Ser Ala Glu His Val Leu Thr Met Leu Asn Glu His Glu Val Lys Phe1 5 10 15Val Asp Leu Arg Phe Thr Asp Thr Lys Gly Lys Glu Gln His Val Thr 20 25 30Ile Pro Ala His Gln Val Asn Ala Glu Phe Phe Glu Glu Gly Lys Met 35 40 45Phe Asp Gly Ser Ser Ile Gly Gly Trp Lys Gly Ile Asn Glu Ser Asp 50 55 60Met Val Leu Met Pro Asp Ala Ser Thr Ala Val Ile Asp Pro Phe Phe65 70 75 80Ala Asp Ser Thr Leu Ile Ile Arg Cys Asp Ile Leu Glu Pro Gly Thr 85 90 95Leu Gln Gly Tyr Asp Arg Asp Pro Arg Ser Ile Ala Lys Arg Ala Glu 100 105 110Asp Tyr Leu Arg Ala Thr Gly Ile Ala Asp Thr Val Leu Phe Gly Pro 115 120 125Glu Pro Glu Phe Phe Leu Phe Asp Asp Ile Arg Phe Gly Ala Ser Ile 130 135 140Ser Gly Ser His Val Ala Ile Asp Asp Ile Glu Gly Ala Trp Asn Ser145 150 155 160Ser Thr Lys Tyr Glu Gly Gly Asn Lys Gly His Arg Pro Gly Val Lys 165 170 175Gly Gly Tyr Phe Pro Val Pro Pro Val Asp Ser Ala Gln Asp Ile Arg 180 185 190Ser Glu Met Cys Leu Val Met Glu Gln Met Gly Leu Val Val Glu Ala 195 200 205His His His Glu Val Ala Thr Ala Gly Gln Asn Glu Val Ala Thr Arg 210 215 220Phe Asn Thr Met Thr Lys Lys Ala Asp Glu Ile Gln Ile Tyr Lys Tyr225 230 235 240Val Val His Asn Val Ala His Arg Phe Gly Lys Thr Ala Thr Phe Met 245 250 255Pro Lys Pro Met Phe Gly Asp Asn Gly Ser Gly Met His Cys His Met 260 265 270Ser Leu Ala Lys Asn Gly Thr Asn Leu Phe Ser Gly Asp Lys Tyr Ala 275 280 285Gly Leu Ser Glu Gln Ala Leu Tyr Tyr Ile Gly Gly Val Ile Lys His 290 295 300Ala Lys Ala Ile Asn Ala Leu Ala Asn Pro Thr Thr Asn Ser Tyr Lys305 310 315 320Arg Leu Val Pro Gly Tyr Glu Ala Pro Val Met Leu Ala Tyr Ser Ala 325 330 335Arg Asn Arg Ser Ala Ser Ile Arg Ile Pro Val Val Ala Ser Pro Lys 340 345 350Ala Arg Arg Ile Glu Val Arg Phe Pro Asp Pro Ala Ala Asn Pro Tyr 355 360 365Leu Cys Phe Ala Ala Leu Leu Met Ala Gly Leu Asp Gly Ile Lys Asn 370 375 380Lys Ile His Pro Gly Glu Pro Met Asp Lys Asn Leu Tyr Asp Leu Pro385 390 395 400Pro Glu Glu Ala Lys Glu Ile Pro Gln Val Ala Gly Ser Leu Glu Glu 405 410 415Ala Leu Asn Ala Leu Asp Leu Asp Arg Glu Phe Leu Lys Ala Gly Gly 420 425 430Val Phe Thr Asp Glu Ala Ile Asp Ala Tyr Ile Ala Leu Arg Arg Glu 435 440 445Glu Asp Asp Arg Val Arg Met Thr Pro His Pro Val Glu Phe Glu Leu 450 455 460Tyr Tyr Ser Val46534468PRTAcidimicrobium ferrooxidans 34Met Gly Tyr Ser Pro Ser Asp Val Val Lys Leu Ile Gln Glu Lys Asp1 5 10 15Ile Lys Phe Ile Asp Phe Arg Phe Thr Asp Thr Lys Gly Lys Glu Gln 20 25 30His Val Ser Val Pro Gly His Val Ile Glu Glu Asp Thr Phe Thr Glu 35 40 45Gly Lys Ala Phe Asp Gly Ser Ser Ile Ala Gly Trp Lys Gly Ile Asn 50 55 60Glu Ser Asp Met Ile Leu Leu Pro Asp Pro Asp Ser Ala Val Leu Asp65 70 75 80Pro Phe Met Asp Glu Thr Thr Leu Leu Leu Arg Cys Asp Val Ile Glu 85 90 95Pro Ala Thr Gly Gln Gly Tyr Glu Arg Asp Pro Arg Ser Val Ala Lys 100 105 110Arg Ala Glu Ile Tyr Leu Lys Ser Thr Gly Ile Ala Asp Thr Ser Phe 115 120 125Phe Gly Pro Glu Phe Phe Val Phe Asp Ser Val Thr Trp Asn Ile Asp 130 135 140Met Ser Gly Cys Ala Tyr Lys Val Asp Ala Glu Glu Ala Ala Trp Asn145 150 155 160Ser Gly Lys Glu Tyr Glu Ser Gly Asn Met Gly His Arg Leu Gly Val 165 170 175Lys Gly Gly Tyr Phe Pro Val Pro Pro Val Asp Ser Ala Gln Asp Leu 180 185 190Arg Ser Ala Met Cys Leu Ala Met Glu Glu Met Gly Leu Lys Val Glu 195 200 205Val His His His Glu Val Ala Thr Ala Gly Gln His Glu Ile Gly Val 210 215 220Gly Phe Asn Thr Leu Thr Pro Arg Arg Met Arg Cys Lys Ile Leu Lys225 230 235 240Tyr Val Val His Asn Val Ala Ala Val Arg Gln Thr Ala Thr Phe Met 245 250 255Pro Lys Pro Val Val Gly Asp Asn Gly Ser Gly Met His Val His Gln 260 265 270Ser Leu Gly Lys Asp Gly Lys Asn Ile Phe Ala Gly Asp Leu Tyr Gly 275 280 285Gly Leu Ser Glu Ile Ala Leu Tyr Tyr Ile Gly Gly Ile Ile Lys His 290 295 300Ala Lys Ala Val Asn Ala Leu Thr Asn Pro Ser Thr Asn Ser Tyr Lys305 310 315 320Arg Leu Val Pro His Phe Glu Ala Pro Val Leu Leu Ala Tyr Ser Ala 325 330 335Lys Asn Arg Ser Ala Ser Ile Arg Ile Pro Tyr Val Asn Ser Pro Lys 340 345 350Ala Arg Arg Ile Glu Val Arg Phe Pro Asp Ser Thr Ala Asn Pro Tyr 355 360 365Leu Ala Phe Ser Ala Met Leu Met Ala Gly Leu Asp Gly Ile Gln Asn 370 375 380Lys Ile His Pro Ala Thr Ala Met Asp Lys Asn Leu Tyr Asp Leu Pro385 390 395 400Ala Glu Glu Gln Ala Asn Ile Pro Gly Val Ala Ala Ser Leu Glu Glu 405 410 415Ala Leu Arg Ala Leu Glu Ala Asp His Asp Phe Leu Met Lys Gly Gly 420 425 430Val Phe Ser Glu Ser Trp Leu Glu Gly Tyr Leu Asp Val Lys Trp Ala 435 440 445Glu Val Gln Thr Leu Arg Val Thr Thr His Pro Val Glu Phe Gln Met 450 455 460Tyr Tyr Ser Leu46535468PRTEscherichia coli 35Ser Ala Glu His Val Leu Thr Met Leu Asn Glu His Glu Val Lys Phe1 5 10 15Val Asp Leu Arg Phe Thr Asp Thr Lys Gly Lys Glu Gln His Val Thr 20 25 30Ile Pro Ala His Gln Val Asn Ala Glu Phe Phe Glu Glu Gly Lys Met 35 40 45Phe Asp Gly Ser Ser Ile Gly Gly Trp Lys Gly Ile Asn Glu Ser Asp 50 55 60Met Val Leu Met Pro Asp Ala Ser Thr Ala Val Ile Asp Pro Phe Phe65 70 75 80Ala Asp Ser Thr Leu Ile Ile Arg Cys Asp Ile Leu Glu Pro Gly Thr 85 90 95Leu Gln Gly Tyr Asp Arg Asp Pro Arg Ser Ile Ala Lys Arg Ala Glu 100 105 110Asp Tyr Leu Arg Ser Thr Gly Ile Ala Asp Thr Val Leu Phe Gly Pro 115 120 125Glu Pro Glu Phe Phe Leu Phe Asp Asp Ile Arg Phe Gly Ser Ser Ile 130 135 140Ser Gly Ser His Val Ala Ile Asp Asp Ile Glu Gly Ala Trp Asn Ser145 150 155 160Ser Thr Gln Tyr Glu Gly Gly Asn Lys Gly His Arg Pro Ala Val Lys 165 170 175Gly Gly Tyr Phe Pro Val Pro Pro Val Asp Ser Ala Gln Asp Ile Arg 180 185 190Ser Glu Met Cys Leu Val Met Glu Gln Met Gly Leu Val Val Glu Ala 195 200 205His His His Glu Val Ala Thr Ala Gly Gln Asn Glu Val Ala Thr Arg 210 215 220Phe Asn Thr Met Thr Lys Lys Ala Asp Glu Ile Gln Ile Tyr Lys Tyr225 230 235 240Val Val His Asn Val Ala His Arg Phe Gly Lys Thr Ala Thr Phe Met 245 250 255Pro Lys Pro Met Phe Gly Asp Asn Gly Ser Gly Met His Cys His Met 260 265 270Ser Leu Ser Lys Asn Gly Val Asn Leu Phe Ala Gly Asp Lys Tyr Ala 275 280 285Gly Leu Ser Glu Gln Ala Leu Tyr Tyr Ile Gly Gly Val Ile Lys His 290 295 300Ala Lys Ala Ile Asn Ala Leu Ala Asn Pro Thr Thr Asn Ser Tyr Lys305 310 315 320Arg Leu Val Pro Gly Tyr Glu Ala Pro Val Met Leu Ala Tyr Ser Ala 325 330 335Arg Asn Arg Ser Ala Ser Ile Arg Ile Pro Val Val Ser Ser Pro Lys 340 345 350Ala Arg Arg Ile Glu Val Arg Phe Pro Asp Pro Ala Ala Asn Pro Tyr 355 360 365Leu Cys Phe Ala Ala Leu Leu Met Ala Gly Leu Asp Gly Ile Lys Asn 370 375 380Lys Ile His Pro Gly Glu Ala Met Asp Lys Asn Leu Tyr Asp Leu Pro385 390 395 400Pro Glu Glu Ala Lys Glu Ile Pro Gln Val Ala Gly Ser Leu Glu Glu 405 410 415Ala Leu Asn Glu Leu Asp Leu Asp Arg Glu Phe Leu Lys Ala Gly Gly 420 425 430Val Phe Thr Asp Glu Ala Ile Asp Ala Tyr Ile Ala Leu Arg Arg Glu 435 440 445Glu Asp Asp Arg Val Arg Met Thr Pro His Pro Val Glu Phe Glu Leu 450 455 460Tyr Tyr Ser Val46536491PRTArchaeoglobus fulgidus 36Met Val Arg Arg Leu Arg Gly Asp Cys Met Glu Glu Val Glu Arg Ala1 5 10 15Lys Ala Val Leu Lys Glu Asn Asn Val Arg Gln Val Leu Cys Ala Phe 20 25 30Ala Asp Val Arg Gly Tyr Leu Gln Met Phe Ser Ile Pro Ala Arg Glu 35 40 45Phe Val Asp Gly Ser Ala Phe Glu Asn Gly Ile Gly Phe Asp Gly Ser 50 55 60Ser Val Arg Gly Phe Arg Thr Ile Glu Lys Ser Asp Met Val Trp Met65 70 75 80Pro Asp Ala Ser Ser Leu Lys Ile Ile Pro Trp Ile Asp Asp Pro Ile 85 90 95Gln Lys Ser Ala Ile Met Phe Gly Asn Val Tyr Glu Ala Trp Gly Thr 100 105 110Glu Ile Ala Asp Cys Asp Pro Arg Gly Tyr Val Ala Lys Arg Tyr Glu 115 120 125Asp Met Leu Lys Ser Glu Gly Met Ser Ala Ile Phe Gly Pro Glu Ile 130 135 140Glu Phe Phe Leu Phe Glu Gly Val Asp Phe Thr Arg Leu Ser Trp Asp145 150 155 160Met Trp Val Ser Pro Asn Gly Gly Ala Gly Asp Ser Trp Gly Pro Pro 165 170 175Arg Ile Met Pro Ile Ser Ser Glu Leu Glu Ser Gly Tyr Met Ile Arg 180 185 190Pro Lys Glu Gly Tyr Phe Arg Pro Pro Pro Glu Asp Thr Thr Val Glu 195 200 205Tyr Arg Asn Glu Leu Val Tyr Tyr Leu Glu Gln Leu Gly Ile Asp Ile 210 215 220Glu Tyr His His His Glu Val Ala Thr Ala Gly Gln Val Glu Leu Asp225 230 235 240Phe Lys Pro Lys Gln Leu Val Asp Val Gly Asp Ala Phe Tyr Leu Tyr 245 250 255Lys Phe Ala Ala Lys Asn Ile Ala Ala Met His Gly Leu Tyr Ala Thr 260 265 270Phe Met Pro Lys Pro Leu Tyr Leu Asp Asn Ala Ser Gly Met His Thr 275 280 285His Gln Ser Leu Trp Lys Gly Glu Pro Phe Ser Gly Glu Ala Val Phe 290 295 300Ala Asp Pro Asp Asp Glu Tyr Met Leu Ser Gln Lys Ala Arg Tyr Tyr305 310 315 320Ile Gly Gly Leu Leu Glu His Ala Lys Ala Leu Thr Ala Leu Cys Ala 325 330 335Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Phe Glu Ala Pro 340 345 350Ile Tyr Ile Cys Trp Ser Pro Arg Asn Arg Ser Ala Leu Val Arg Val 355 360 365Pro Met Tyr Val Lys Lys Pro Ser Ala Ile Arg Val Glu Tyr Arg Gly 370 375 380Val Asp Pro Ser Cys Asn Pro Tyr Leu Ala Ile Thr Ala Gln Leu Ala385 390 395 400Ala Gly Leu Asp Gly Ile Lys Lys Lys Ile Asp Pro Gly Asp Pro Leu 405 410 415Leu Glu Asp Val Tyr Glu Leu Thr Pro Ala Gln Lys Arg Glu Leu Gly 420 425 430Val Gly Glu Leu Pro Thr Thr Leu Arg Asp Ala Ile Asp His Leu Ala 435 440 445Ser Asp Glu Leu Met Gln Glu Val Leu Gly Ser His Ile Phe Asp Ala 450 455 460Phe Met Glu Leu Lys Ile Asp Glu Trp Asn Gln Tyr Cys Leu Tyr Ile465 470 475 480Thr Pro Trp Glu Phe Met Lys Tyr Phe Asp Ile 485 49037467PRTAzotobacter vinelandii 37Met Ser Lys Ser Leu Gln Leu Ile Lys Glu His Asp Val Lys Trp Ile1 5 10 15Asp Leu Arg Phe Thr Asp Thr Lys Gly Lys Gln Gln His Val Thr Met 20 25 30Pro Ala Arg Asp Val Asp Asp Asp Phe Phe Glu Tyr Gly Lys Met Phe 35

40 45Asp Gly Ser Ser Ile Ala Gly Trp Lys Gly Ile Glu Ala Ser Asp Met 50 55 60Ile Leu Met Pro Asp Asp Ser Thr Ala Val Leu Asp Pro Phe Thr Glu65 70 75 80Glu Pro Thr Leu Ile Ile Val Cys Asp Ile Ile Glu Pro Ser Thr Met 85 90 95Gln Gly Tyr Asp Arg Asp Pro Arg Ala Ile Ala Arg Arg Ala Glu Glu 100 105 110Tyr Leu Lys Ser Thr Gly Ile Gly Asp Thr Ala Phe Phe Gly Pro Glu 115 120 125Pro Glu Phe Phe Ile Phe Asp Glu Val Lys Tyr Lys Ser Asp Ile Ser 130 135 140Gly Ser Met Phe Lys Ile Phe Ser Glu Gln Ala Ala Trp Asn Thr Asp145 150 155 160Ala Asp Phe Glu Gly Gly Asn Lys Gly His Arg Pro Gly Val Lys Gly 165 170 175Gly Tyr Phe Pro Val Pro Pro Val Asp His Asp His Glu Ile Arg Thr 180 185 190Ala Met Cys Asn Ala Leu Glu Glu Met Gly Leu Lys Val Glu Val His 195 200 205His His Glu Val Ala Thr Ala Gly Gln Asn Glu Ile Gly Val Ser Phe 210 215 220Asn Thr Leu Val Ala Lys Ala Asp Glu Val Gln Thr Leu Lys Tyr Cys225 230 235 240Val His Asn Val Ala Asp Ala Tyr Gly Lys Thr Val Thr Phe Met Pro 245 250 255Lys Pro Leu Tyr Gly Asp Asn Gly Ser Gly Met His Val His Met Ser 260 265 270Ile Ala Lys Asp Gly Lys Asn Thr Phe Ala Gly Glu Gly Tyr Ala Gly 275 280 285Leu Ser Asp Thr Ala Leu Tyr Phe Ile Gly Gly Ile Ile Lys His Gly 290 295 300Lys Ala Leu Asn Gly Phe Thr Asn Pro Ser Thr Asn Ser Tyr Lys Arg305 310 315 320Leu Val Pro Gly Phe Glu Ala Pro Val Met Leu Ala Tyr Ser Ala Arg 325 330 335Asn Arg Ser Ala Ser Ile Arg Ile Pro Tyr Val Asn Ser Pro Lys Ala 340 345 350Arg Arg Ile Glu Ala Arg Phe Pro Asp Pro Ser Ala Asn Pro Tyr Leu 355 360 365Ala Phe Ala Ala Leu Leu Met Ala Gly Leu Asp Gly Ile Gln Asn Lys 370 375 380Ile His Pro Gly Asp Ala Ala Asp Lys Asn Leu Tyr Asp Leu Pro Pro385 390 395 400Glu Glu Ala Lys Glu Ile Pro Gln Val Cys Gly Ser Leu Lys Glu Ala 405 410 415Leu Glu Glu Leu Asp Lys Gly Arg Ala Phe Leu Thr Lys Gly Gly Val 420 425 430Phe Ser Asp Asp Phe Ile Asp Ala Tyr Leu Glu Leu Lys Ser Glu Glu 435 440 445Glu Ile Lys Val Arg Thr Phe Val His Pro Leu Glu Tyr Asp Leu Tyr 450 455 460Tyr Ser Val46538443PRTBacillus cereus 38Ala Arg Tyr Thr Lys Glu Asp Ile Phe Arg Leu Ala Lys Glu Glu Asn1 5 10 15Val Lys Tyr Ile Arg Leu Gln Phe Thr Asp Leu Leu Gly Val Ile Lys 20 25 30Asn Val Glu Ile Pro Val Ser Gln Leu Thr Lys Ala Leu Asp Asn Lys 35 40 45Met Met Phe Asp Gly Ser Ser Ile Glu Gly Phe Val Arg Ile Glu Glu 50 55 60Ser Asp Met Tyr Leu Tyr Pro Asp Leu Asp Thr Trp Val Ile Phe Pro65 70 75 80Trp Thr Ala Glu Lys Gly Lys Val Ala Arg Leu Ile Cys Asp Ile Tyr 85 90 95Asn Ala Asp Gly Thr Pro Phe Glu Gly Asp Pro Arg Asn Asn Leu Lys 100 105 110Arg Val Leu Lys Glu Met Glu Ala Leu Gly Phe Ser Asp Phe Asn Leu 115 120 125Gly Pro Glu Pro Glu Phe Phe Leu Phe Lys Val Asp Glu Lys Gly Asn 130 135 140Pro Thr Leu Glu Leu Asn Asp Asn Gly Gly Tyr Phe Asp Leu Ala Pro145 150 155 160Met Asp Leu Gly Glu Asn Cys Arg Arg Asp Ile Val Leu Glu Leu Glu 165 170 175Glu Met Gly Phe Glu Ile Glu Ala Ser His His Glu Val Ala Pro Gly 180 185 190Gln His Glu Ile Asp Phe Lys Tyr Ala Asn Ala Ile Arg Ser Cys Asp 195 200 205Asp Ile Gln Thr Phe Lys Leu Val Val Lys Thr Ile Ala Arg Lys His 210 215 220Gly Leu His Ala Thr Phe Met Pro Lys Pro Leu Tyr Gly Val Asn Gly225 230 235 240Ser Gly Met His Cys Asn Leu Ser Leu Phe Lys Asn Gly Glu Asn Val 245 250 255Phe Tyr Asp Gln Asn Gly Asp Leu Gln Leu Ser Asp Asp Ala Arg His 260 265 270Phe Ile Ala Gly Ile Leu Lys His Ala Pro Ala Phe Thr Ala Val Ala 275 280 285Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu Ala 290 295 300Pro Cys Tyr Val Ala Trp Ser Ala Gln Asn Arg Ser Pro Leu Val Arg305 310 315 320Ile Pro Ala Ser Arg Gly Ile Ser Thr Arg Val Glu Val Arg Ser Val 325 330 335Asp Pro Ala Ala Asn Pro Tyr Leu Val Met Ala Thr Leu Leu Ala Ala 340 345 350Gly Leu Asp Gly Ile Lys Asn Lys Leu Thr Pro Pro Ala Ala Val Asp 355 360 365Arg Asn Ile Tyr Val Met Thr Lys Glu Glu Arg Glu Glu Ala Gly Ile 370 375 380Val Asp Leu Pro Ala Thr Leu Ala Gln Ala Leu Val Thr Leu Gln Ser385 390 395 400Asn Glu Val Ile Ser Asn Ala Leu Gly Asp His Leu Leu Glu His Phe 405 410 415Ile Glu Ala Lys Glu Phe Glu Trp Asp Ile Phe Arg Thr Gln Val His 420 425 430Gln Trp Glu Arg Asp Gln Tyr Met Ser Leu Tyr 435 44039443PRTBacillus subtilis 39Ala Lys Tyr Thr Arg Glu Asp Ile Glu Lys Leu Val Lys Glu Glu Asn1 5 10 15Val Lys Tyr Ile Arg Leu Gln Phe Thr Asp Ile Leu Gly Thr Ile Lys 20 25 30Asn Val Glu Ile Pro Val Ser Gln Leu Gly Lys Ala Leu Asp Asn Lys 35 40 45Val Met Phe Asp Gly Ser Ser Ile Glu Gly Phe Val Arg Ile Glu Glu 50 55 60Ser Asp Met Tyr Leu Tyr Pro Asp Leu Asn Thr Phe Val Ile Phe Pro65 70 75 80Trp Thr Ala Glu Lys Gly Lys Val Ala Arg Phe Ile Cys Asp Ile Tyr 85 90 95Asn Pro Asp Gly Thr Pro Phe Glu Gly Asp Pro Arg Asn Asn Leu Lys 100 105 110Arg Ile Leu Lys Glu Met Glu Asp Leu Gly Phe Ser Asp Phe Asn Leu 115 120 125Gly Pro Glu Pro Glu Phe Phe Leu Phe Lys Leu Asp Glu Lys Gly Glu 130 135 140Pro Thr Leu Glu Leu Asn Asp Lys Gly Gly Tyr Phe Asp Leu Ala Pro145 150 155 160Thr Asp Leu Gly Glu Asn Cys Arg Arg Asp Ile Val Leu Glu Leu Glu 165 170 175Glu Met Gly Phe Glu Ile Glu Ala Ser His His Glu Val Ala Pro Gly 180 185 190Gln His Glu Ile Asp Phe Lys Tyr Ala Gly Ala Val Arg Ser Cys Asp 195 200 205Asp Ile Gln Thr Phe Lys Leu Val Val Lys Thr Ile Ala Arg Lys His 210 215 220Gly Leu His Ala Thr Phe Met Pro Lys Pro Leu Phe Gly Val Asn Gly225 230 235 240Ser Gly Met His Cys Asn Leu Ser Leu Phe Lys Asn Gly Val Asn Ala 245 250 255Phe Phe Asp Glu Asn Ala Asp Leu Gln Leu Ser Glu Thr Ala Lys His 260 265 270Phe Ile Ala Gly Ile Val Lys His Ala Thr Ser Phe Thr Ala Val Thr 275 280 285Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Val Pro Gly Tyr Glu Ala 290 295 300Pro Cys Tyr Val Ala Trp Ser Ala Gln Asn Arg Ser Pro Leu Ile Arg305 310 315 320Ile Pro Ala Ser Arg Gly Ile Ser Thr Arg Val Glu Val Arg Ser Val 325 330 335Asp Pro Ala Ala Asn Pro Tyr Leu Ala Leu Ser Val Leu Leu Ala Ala 340 345 350Gly Leu Asp Gly Ile Lys Asn Lys Leu Glu Ala Pro Ala Pro Ile Asp 355 360 365Arg Asn Ile Tyr Val Met Ser Lys Glu Glu Arg Met Glu Asn Gly Ile 370 375 380Val Asp Leu Pro Ala Thr Leu Ala Glu Ala Leu Glu Glu Phe Lys Ser385 390 395 400Asn Glu Val Met Val Lys Ala Leu Gly Glu His Leu Phe Glu His Phe 405 410 415Ile Glu Ala Lys Glu Ile Glu Trp Asp Met Phe Arg Thr Gln Val His 420 425 430Pro Trp Glu Arg Glu Gln Tyr Met Ser Gln Tyr 435 44040454PRTHalobacterium volcanii 40Met Thr Glu Asp Asn Ala Leu Thr Asp Gly Gly Leu Ser Asp Glu Ala1 5 10 15Gln Ala Val Ile Asp Glu Ile Glu Glu Lys Asn Val Asp Phe Leu Arg 20 25 30Leu Gln Phe Thr Asp Ile Leu Gly Thr Val Lys Asn Val Ser Ile Pro 35 40 45Ala Ser Gln Ala Glu Lys Ala Phe Thr Glu Gly Ile Tyr Phe Asp Gly 50 55 60Ser Ser Ile Asp Gly Phe Val Arg Ile Gln Glu Ser Asp Met Arg Leu65 70 75 80Glu Pro Asp Pro Ser Thr Phe Ala Val Leu Pro Trp Arg Lys Lys Glu 85 90 95Asn Ser Ala Ala Gly Arg Leu Ile Cys Asp Val Phe Asn Thr Ser Thr 100 105 110Gly Glu Pro Phe Ser Gly Asp Pro Arg Gly Val Leu Lys Arg Ala Ile 115 120 125Glu Pro Glu Glu Leu Gly Tyr Asp Val Asn Val Ala Pro Glu Pro Glu 130 135 140Phe Phe Leu Phe Glu Glu Asp Glu Asp Gly Arg Ala Thr Thr Val Thr145 150 155 160Asn Asp Ala Gly Gly Tyr Phe Asp Leu Ala Pro Lys Asp Leu Ala Ser 165 170 175Asp Val Arg Arg Asp Ile Ile Tyr Gly Leu Glu Ser Met Gly Phe Asp 180 185 190Ile Glu Ala Ser His His Glu Val Ala Glu Gly Gln His Glu Ile Asn 195 200 205Phe Thr Tyr Asp Asp Ala Leu Ser Thr Ala Asp Asn Val Arg Thr Phe 210 215 220Arg Ser Val Val Arg Ala Ile Ala Ala Glu His Asp Leu His Ala Thr225 230 235 240Phe Met Pro Lys Pro Ile Pro Arg Ile Asn Gly Ser Gly Met His Thr 245 250 255His Ile Ser Leu Phe Lys Asp Gly Glu Asn Ala Phe His Asp Gly Asp 260 265 270Asp Glu Phe Asp Leu Ser Asp Thr Ala Lys Ser Phe Val Ala Gly Ile 275 280 285Leu Asp His Ala Pro Ala Ile Thr Ala Ser Leu Thr Arg Arg Ser Thr 290 295 300Pro Thr Arg Arg Leu Val Pro Gly Tyr Glu Ala Pro Val Tyr Ile Ala305 310 315 320Trp Ser Asp Arg Asn Arg Ser Ala Leu Ile Arg Lys Pro Ala Ala Arg 325 330 335Thr Pro Ala Ala Ser Arg Ile Glu Ala Arg Phe Pro Asp Pro Ser Cys 340 345 350Asn Pro Tyr Leu Ala Phe Ala Ala Leu Ile His Ala Gly Leu Asp Gly 355 360 365Val Glu Lys Gly Leu Asp Cys Pro Asp Pro Val Arg Glu Asn Ile Tyr 370 375 380Glu Phe Asp Glu Ala Lys Arg Glu Glu Tyr Gly Ile Glu Thr Leu Pro385 390 395 400Lys Thr Ser Ala Ala Arg Arg Arg Pro Arg Arg Asp Glu Val Ile Gln 405 410 415Glu Ala Leu Gly Asp His Val Phe Glu Lys Phe Val Glu Ala Lys Arg 420 425 430Ser Glu Phe Lys Asp Tyr Leu Val Asp Val Ser Gln Trp Glu Leu Asp 435 440 445Arg Tyr Leu Glu Thr Phe 45041445PRTLactobacillus delbrueckii 41Met Ser Lys Val Ile Thr Glu Glu Glu Ile Arg Lys Asp Val Glu Glu1 5 10 15Lys Asn Val Arg Phe Leu Arg Leu Ala Phe Thr Asp Ile Asn Gly Thr 20 25 30Leu Lys Asn Leu Glu Val Pro Val Ser Gln Leu Asp Asp Val Leu Gly 35 40 45Asn Gln Thr Arg Phe Asp Gly Ser Ser Ile Asp Gly Phe Val Arg Leu 50 55 60Glu Glu Ser Asp Met Val Leu Tyr Pro Asp Leu Ala Thr Trp Leu Val65 70 75 80Leu Ala Trp Thr Thr Val Glu Glu Gly Thr Ile Gly Arg Leu Val Cys 85 90 95Ser Val His Asn Val Asp Gly Thr Pro Phe Glu Gly Asp Pro Arg Asn 100 105 110Asn Leu Lys Lys Val Ile Ala Glu Met Glu Glu Met Gly Phe Ser Asp 115 120 125Phe Glu Ile Gly Phe Glu Ala Glu Phe Phe Leu Phe Lys Glu Gly Lys 130 135 140Asn Gly Glu Glu Thr Thr Lys Val Ser Asp His Ser Ser Tyr Phe Asp145 150 155 160Met Ala Ser Glu Asp Glu Gly Ala Lys Cys Arg Arg Glu Ile Val Glu 165 170 175Thr Leu Glu Lys Leu Gly Phe Arg Val Glu Ala Ala His His Glu Val 180 185 190Gly Asp Gly Gln Gln Glu Ile Asp Phe Arg Phe Asp Asn Ala Leu Ala 195 200 205Thr Ala Asp Lys Leu Gln Thr Phe Lys Met Val Val Lys Thr Ile Ala 210 215 220Arg Lys Tyr His Leu His Ala Ser Phe Met Ala Lys Pro Val Glu Gly225 230 235 240Leu Ala Gly Asn Gly Met His Thr Asn Met Ser Leu Leu Lys Asp Gly 245 250 255Lys Asn Ala Phe Tyr Asp Lys Asp Gly Gln Tyr Asn Leu Ser Thr Thr 260 265 270Ala Leu Thr Phe Leu Asn Gly Ile Leu Glu His Ala Arg Ala Ile Thr 275 280 285Cys Val Ala Asn Pro Thr Val Asn Ser Tyr Lys Arg Leu Ile Pro Gly 290 295 300Phe Glu Ala Pro Val Tyr Ile Ser Trp Ala Ser Arg Asn Arg Ser Pro305 310 315 320Met Val Arg Ile Pro Asn Ala Asn Glu Val Gly Thr Arg Leu Glu Met 325 330 335Arg Ser Thr Asp Pro Thr Ala Asn Pro Tyr Leu Leu Leu Ser Ala Cys 340 345 350Leu Lys Ala Gly Leu Thr Gly Ile Lys Glu Gly Lys Leu Pro Met Ala 355 360 365Pro Val Thr Ser Asn Leu Phe Glu Met Thr Asp Asp Glu Arg Lys Glu 370 375 380Leu Gly Ile Lys Pro Leu Pro Ser Thr Leu His Asn Ala Ile Lys Ala385 390 395 400Phe Lys Glu Asp Glu Val Val Lys Ser Ala Phe Ser Glu His Ile Val 405 410 415Asp Ser Phe Leu Glu Leu Lys Glu Thr Glu Trp Ala Leu Tyr Thr Gln 420 425 430Ser Val Ser Glu Trp Glu Val Lys Arg Tyr Phe Asn Tyr 435 440 44542428PRTPlasmodium falciparum 42Met Cys Asp Ile Lys Arg Tyr Asn Gly Phe Asp Tyr Tyr Lys Cys Pro1 5 10 15Arg Thr Ile Leu Lys Lys Thr Cys Glu Phe Val Lys Asn Glu Gly Ile 20 25 30Ala Asp Lys Val Cys Ile Gly Asn Glu Leu Glu Phe Phe Ile Phe Asp 35 40 45Lys Val Asn Tyr Ser Leu Asp Glu Tyr Asn Thr Tyr Leu Lys Val Tyr 50 55 60Asp Arg Glu Ser Phe Ser Cys Lys Asn Asp Leu Ser Ser Ile Tyr Gly65 70 75 80Asn His Val Val Asn Lys Val Glu Pro His Lys Asp His Phe Asn Asn 85 90 95Pro Asn Asn Glu Tyr Leu Ile Asn Asp Asp Ser Lys Lys Val Lys Lys 100 105 110Lys Ser Gly Tyr Phe Thr Thr Asp Pro Tyr Asp Thr Ser Asn Ile Ile 115 120 125Lys Leu Arg Ile Cys Arg Ala Leu Asn Asp Met Asn Ile Asn Val Gln 130 135 140Arg Tyr His His Glu Val Ser Thr Ser Gln His Glu Ile Ser Leu Lys145 150 155 160Tyr Phe Asp Ala Leu Thr Asn Ala Asp Phe Leu Leu Ile Thr Lys Gln 165 170 175Ile Ile Lys Thr Thr Val Ser Ser Phe Asn Arg Thr Ala Thr Phe Met 180 185 190Pro Lys Pro Leu Val Asn Asp Asn Gly Asn Gly Leu His Cys Asn Ile 195 200 205Ser Leu Trp Lys Asn Asn Lys Asn Ile Phe Tyr His Asn Asp Pro Ser 210 215 220Thr Phe Phe Leu Ser Lys Glu Ser Phe Tyr Phe Met Tyr Gly Ile Val225 230 235 240Lys His Ala Lys Ala Leu Gln Ala Phe Cys Asn Ala Thr Met Asn Ser 245

250 255Tyr Lys Arg Leu Val Pro Gly Phe Glu Thr Cys Gln Lys Leu Phe Tyr 260 265 270Ser Phe Gly Ser Arg Ser Ala Val Ile Arg Leu Ser Leu Ile Asn Tyr 275 280 285Ser Asn Pro Ser Glu Lys Arg Ile Glu Phe Arg Leu Pro Asp Cys Ala 290 295 300Asn Ser Pro His Leu Val Met Ala Ala Ile Ile Leu Ala Gly Tyr Asp305 310 315 320Gly Ile Lys Ser Lys Glu Gln Pro Leu Val Pro Phe Glu Ser Lys Asp 325 330 335Asn His Phe Tyr Ile Ser Ser Ile Phe Ser Lys Tyr Val Gln His Pro 340 345 350Glu Asn Phe Asn Ile Leu Thr His Ala Leu Glu Gly Tyr Glu Ser Leu 355 360 365His Thr Ile Asn Glu Ser Pro Glu Phe Lys Asn Phe Phe Lys Cys Glu 370 375 380Glu Pro Gln Gly Ile Ser Phe Ser Leu Val Glu Ser Leu Asp Ala Leu385 390 395 400Glu Lys Asp His Ala Phe Leu Thr Val Asn Asn Ile Phe Thr Glu Val 405 410 415Ser Gln Arg Ile Lys Asn Lys His Tyr His Gly Lys 420 42543369PRTSaccharomyces cerevisiae 43Ala Glu Ala Ser Ile Glu Lys Thr Gln Ile Leu Gln Lys Tyr Leu Glu1 5 10 15Leu Asp Gln Arg Gly Arg Ile Ile Ala Glu Tyr Val Trp Ile Asp Gly 20 25 30Thr Gly Asn Leu Arg Ser Lys Gly Arg Thr Leu Lys Lys Arg Ile Thr 35 40 45Ser Ile Asp Gln Leu Pro Glu Trp Asn Phe Asp Gly Ser Ser Thr Asn 50 55 60Gln Ala Pro Gly His Asp Ser Asp Ile Tyr Leu Lys Pro Val Ala Tyr65 70 75 80Tyr Pro Asp Pro Phe Arg Arg Gly Asp Asn Ile Val Val Leu Ala Ala 85 90 95Cys Tyr Asn Asn Asp Gly Thr Pro Asn Lys Phe Asn His Arg His Glu 100 105 110Ala Ala Lys Leu Phe Ala Ala His Lys Asp Glu Glu Ile Trp Phe Gly 115 120 125Leu Glu Gln Glu Tyr Thr Leu Phe Asp Met Tyr Asp Asp Val Tyr Gly 130 135 140Trp Pro Lys Gly Gly Tyr Pro Ala Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155 160Val Gly Ala Gly Lys Val Tyr Ala Arg Asp Met Ile Glu Ala His Tyr 165 170 175Arg Ala Cys Leu Tyr Ala Gly Leu Glu Ile Ser Gly Ile Asn Ala Glu 180 185 190Val Met Pro Ser Gln Trp Glu Phe Gln Val Gly Pro Cys Thr Gly Ile 195 200 205Asp Met Gly Asp Gln Leu Trp Met Ala Arg Tyr Phe Leu His Arg Val 210 215 220Ala Glu Glu Phe Gly Ile Lys Ile Ser Phe His Pro Lys Pro Leu Lys225 230 235 240Gly Asp Trp Asn Gly Ala Gly Cys His Thr Asn Val Ser Thr Lys Glu 245 250 255Met Arg Gln Pro Gly Gly Met Lys Tyr Ile Glu Gln Ala Ile Glu Lys 260 265 270Leu Ser Lys Arg His Ala Glu His Ile Lys Leu Tyr Gly Ser Asp Asn 275 280 285Asp Met Arg Leu Thr Gly Arg His Glu Thr Ala Ser Met Thr Ala Phe 290 295 300Ser Ser Gly Val Ala Asn Arg Gly Ser Ser Ile Arg Ile Pro Arg Ser305 310 315 320Val Ala Lys Glu Gly Tyr Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Ile Asp Pro Tyr Leu Val Thr Gly Ile Met Cys Glu Thr Val Cys 340 345 350Gly Ala Ile Asp Asn Ala Asp Met Thr Lys Glu Phe Glu Arg Glu Ser 355 360 365Ser44382PRTChlamydomonas reinhardtii 44Met Ala Ala Gly Ser Val Gly Val Phe Ala Thr Asp Glu Lys Ile Gly1 5 10 15Ser Leu Leu Asp Gln Ser Ile Thr Arg His Phe Leu Ser Thr Val Thr 20 25 30Asp Gln Gln Gly Lys Ile Cys Ala Glu Tyr Val Trp Ile Gly Gly Ser 35 40 45Met His Asp Val Arg Ser Lys Ser Arg Thr Leu Ser Thr Ile Pro Thr 50 55 60Lys Pro Glu Asp Leu Pro His Trp Asn Tyr Asp Gly Ser Ser Thr Gly65 70 75 80Gln Ala Pro Gly His Asp Ser Glu Val Tyr Leu Ile Pro Arg Ser Ile 85 90 95Phe Lys Asp Pro Phe Arg Gly Gly Asp Asn Ile Leu Val Met Cys Asp 100 105 110Cys Tyr Glu Pro Pro Lys Val Asn Pro Asp Gly Thr Leu Ala Ala Pro 115 120 125Lys Pro Ile Pro Thr Asn Thr Arg Phe Ala Cys Ala Glu Val Met Glu 130 135 140Lys Ala Lys Lys Glu Glu Pro Trp Phe Gly Ile Glu Gln Glu Tyr Thr145 150 155 160Leu Leu Asn Ala Ile Thr Lys Trp Pro Leu Gly Trp Pro Lys Gly Gly 165 170 175Tyr Pro Ala Pro Gln Gly Pro Tyr Tyr Cys Ser Ala Gly Ala Gly Val 180 185 190Ala Ile Gly Arg Asp Val Ala Glu Val His Tyr Arg Leu Cys Leu Ala 195 200 205Ala Gly Val Asn Ile Ser Gly Val Asn Ala Glu Val Leu Pro Ser Gln 210 215 220Trp Glu Tyr Gln Val Gly Pro Cys Glu Gly Ile Thr Met Gly Asp His225 230 235 240Met Trp Met Ser Arg Tyr Ile Met Tyr Arg Val Cys Glu Met Phe Asn 245 250 255Val Glu Val Ser Phe Asp Pro Lys Pro Ile Pro Gly Asp Trp Asn Gly 260 265 270Ser Gly Gly His Thr Asn Tyr Ser Thr Lys Ala Thr Arg Thr Ala Pro 275 280 285Asp Gly Trp Lys Val Ile Gln Glu His Cys Ala Lys Leu Glu Ala Arg 290 295 300His Ala Val His Ile Ala Ala Tyr Gly Glu Gly Asn Glu Arg Arg Leu305 310 315 320Thr Gly Lys His Glu Thr Ser Ser Met Ser Asp Phe Ser Trp Gly Val 325 330 335Ala Asn Arg Gly Cys Ser Ile Arg Val Gly Arg Met Val Pro Val Glu 340 345 350Lys Ser Gly Tyr Tyr Glu Asp Arg Arg Pro Ala Ser Asn Leu Asp Ala 355 360 365Tyr Val Val Thr Arg Leu Ile Val Glu Thr Thr Ile Leu Leu 370 375 38045357PRTZea mays 45Met Ala Ser Leu Thr Asp Leu Val Asn Leu Asp Leu Ser Asp Cys Thr1 5 10 15Asp Arg Ile Ile Ala Glu Tyr Ile Trp Ile Gly Gly Thr Gly Ile Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile Thr Asp Pro Ile 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Lys Gly Asn His Ile Leu Val Met Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg Tyr Ser Ala Ala Lys 100 105 110Val Phe Ser His Pro Asp Val Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Ser Trp Pro Leu Gly 130 135 140Trp Pro Val Gly Gly Tyr Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155 160Ala Gly Ala Asp Lys Ala Phe Gly Arg Asp Val Val Asp Ala His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Met Ala Gly Ile Val Leu Ser Leu Asp Pro Lys Pro Ile Lys225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Glu Ala Gly Gly Tyr Glu Val Ile Lys Ala Ala Ile Asp Lys 260 265 270Leu Gly Lys Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Lys Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Arg Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Gly Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Asn Gly Asn 35546357PRTOrysa sativa 46Met Ala Asn Leu Thr Asp Leu Val Asn Leu Asn Leu Ser Asp Cys Ser1 5 10 15Asp Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Val Lys Gly Pro Ile Thr Asp Val Ser 35 40 45Gln Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Glu Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asp Asn Ile Leu Val Met Cys Asp Cys Tyr Thr 85 90 95Pro Gln Gly Glu Pro Ile Pro Thr Asn Lys Arg His Ser Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Val Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Val Asn Trp Pro Leu Gly 130 135 140Trp Pro Val Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Ala145 150 155 160Ala Gly Ala Glu Lys Ala Phe Gly Arg Asp Ile Val Asp Ala His Tyr 165 170 175Lys Ala Cys Ile Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ala Ala Ala Asp Gln Val Trp Val Ala Arg Tyr Ile Leu Glu Arg Val 210 215 220Thr Glu Val Ala Gly Val Val Leu Ser Leu Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Phe Ser Thr Lys Ser 245 250 255Met Arg Glu Pro Gly Gly Tyr Glu Val Ile Lys Lys Ala Ile Asp Lys 260 265 270Leu Ala Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Asn Thr Phe 290 295 300Lys Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Gly Met Ile Ala Glu Thr Thr Leu 340 345 350Leu Trp Lys Gln Asn 35547353PRTLupinus luteus 47Met Ser Val Leu Ser Asp Leu Ile Asn Leu Asn Leu Ser Asp Thr Thr1 5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Val Asp 20 25 30Leu Arg Ser Lys Ala Arg Thr Leu Ser Gly Pro Val Asn Asp Pro Ser 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Gly Gln Ala Pro 50 55 60Gly Lys Asp Ser Glu Val Ile Leu Trp Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Met Cys Asp Thr Tyr Thr 85 90 95Pro Ala Gly Glu Pro Ile Pro Thr Asn Lys Arg His Ala Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Val Ala Glu Glu Pro Trp Phe Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile His Trp Pro Ile Gly 130 135 140Trp Pro Leu Gly Gly Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys Gly145 150 155 160Thr Gly Ala Glu Lys Ala Phe Gly Arg Asp Ile Val Asp Ser His Tyr 165 170 175Lys Ala Cys Leu Tyr Ala Gly Ile Asn Ile Ser Gly Ile Asn Ala Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Leu Trp Val Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Leu Ser Leu Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Thr Asn Tyr Ser Thr Lys Ser 245 250 255Met Arg Asn Asp Gly Gly Tyr Glu Val Ile Lys Lys Ala Ile Glu Lys 260 265 270Leu Gly Lys Arg His Asn Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Arg His Glu Thr Ala Asp Ile Ser Thr Phe 290 295 300Phe Trp Gly Val Ala Asn Arg Gly Ala Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Glu Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Leu 340 345 350Leu48357PRTPisum sativum 48Met Ser Ser Leu Ser Asp Leu Ile Asn Phe Asn Leu Ser Asp Ser Thr1 5 10 15Glu Lys Ile Ile Ala Glu Tyr Ile Trp Val Gly Gly Ser Gly Ile Asp 20 25 30Ile Arg Ser Lys Ala Arg Thr Leu Pro Gly Pro Val Ser Asp Pro Ala 35 40 45Lys Leu Pro Lys Trp Asn Tyr Asp Gly Ser Ser Thr Asn Gln Ala Pro 50 55 60Gly Lys Asp Ser Glu Val Ile Leu Tyr Pro Gln Ala Ile Phe Lys Asp65 70 75 80Pro Phe Arg Arg Gly Asn Asn Ile Leu Val Ile Cys Asp Val Tyr Thr 85 90 95Pro Ala Gly Glu Pro Leu Pro Thr Asn Lys Arg Tyr Asn Ala Ala Lys 100 105 110Ile Phe Ser His Pro Asp Val Ala Ala Glu Val Pro Trp Tyr Gly Ile 115 120 125Glu Gln Glu Tyr Thr Leu Leu Gln Lys Asp Ile Asn Trp Pro Leu Gly 130 135 140Trp Pro Ile Gly Gly Tyr Pro Gly Lys Gln Gly Pro Tyr Tyr Cys Gly145 150 155 160Ile Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val Asp Ala His Tyr 165 170 175Lys Ala Cys Leu Phe Ala Gly Ile Asn Ile Ser Gly Ile Asn Gly Glu 180 185 190Val Met Pro Gly Gln Trp Glu Phe Gln Val Gly Pro Ser Val Gly Ile 195 200 205Ser Ala Gly Asp Glu Ile Trp Ala Ala Arg Tyr Ile Leu Glu Arg Ile 210 215 220Thr Glu Ile Ala Gly Val Val Val Ser Phe Asp Pro Lys Pro Ile Pro225 230 235 240Gly Asp Trp Asn Gly Ala Gly Ala His Ala Asn Phe Ser Thr Lys Ser 245 250 255Met Arg Glu Asn Gly Gly Tyr Glu Val Ile Lys Lys Ala Ile Glu Lys 260 265 270Leu Gly Leu Arg His Lys Glu His Ile Ala Ala Tyr Gly Glu Gly Asn 275 280 285Glu Arg Arg Leu Thr Gly Lys His Glu Thr Ala Asp Ile Asn Val Phe 290 295 300Ser Trp Gly Val Ala Asn Arg Gly Ser Ser Ile Arg Val Gly Arg Asp305 310 315 320Thr Glu Lys Asp Gly Lys Gly Tyr Phe Glu Asp Arg Arg Pro Ala Ser 325 330 335Asn Met Asp Pro Tyr Val Val Thr Ser Met Ile Ala Glu Thr Thr Ile 340 345 350Leu Trp Lys Lys Pro 35549399PRTDrosophila melanogaster 49Met Ala Leu Arg Val Ala Gly Leu Phe Leu Lys Lys Glu Leu Val Ala1 5 10 15Pro Ala Thr Gln Gln Leu Arg Leu Leu Arg Thr Gly Asn Thr Thr Arg 20 25 30Ser Gln Phe Leu Ala Asn Ser Pro Asn Thr Ala Leu Asp Lys Ser Ile 35 40 45Leu Gln Arg Tyr Arg Asn Leu Glu Thr Pro Ala Asn Arg Val Gln Ala 50 55 60Thr Tyr Leu Trp Ile Asp Gly Thr Gly Glu Asn Ile Arg Leu Lys Asp65 70 75 80Arg Val Leu Asp Lys Val Pro Ser Ser Val Glu Asp Leu Pro Asp Trp 85 90 95Gln Tyr

Asp Gly Ser Ser Thr Tyr Gln Ala His Gly Glu Asn Ser Asp 100 105 110Thr Thr Leu Lys Pro Arg Ala Ile Tyr Arg Asp Pro Phe Lys Pro Gly 115 120 125Lys Asn Asp Val Ile Val Leu Cys Asp Thr Tyr Ser Ala Asp Gly Lys 130 135 140Pro Thr Ala Ser Asn Lys Arg Ala Ala Phe Gln Ala Ala Ile Asp Leu145 150 155 160Ile Ser Asp Gln Glu Pro Trp Phe Gly Ile Glu Gln Glu Tyr Thr Leu 165 170 175Leu Arg Arg Gly Arg Thr Ser Phe Gly Trp Pro Glu Asn Gly Phe Pro 180 185 190Ala Pro Gln Gly Pro Tyr Tyr Cys Gly Val Gly Ala Asp Arg Val Tyr 195 200 205Ala Arg Asp Leu Val Glu Ala His Val Val Ala Cys Leu Tyr Ala Gly 210 215 220Ile Asp Phe Ala Gly Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu225 230 235 240Phe Gln Ile Gly Pro Ala Gly Ile Lys Ala Cys Asp Asp Leu Trp Val 245 250 255Ser Arg Tyr Ile Leu Gln Arg Ile Ala Glu Glu Tyr Gly Val Val Val 260 265 270Thr Phe Asp Pro Lys Pro Met Glu Gly Gln Trp Asn Gly Ala Gly Arg 275 280 285His Thr Asn Phe Ser Thr Lys Glu Met Arg Ala Asp Gly Gly Ile Lys 290 295 300Ala Ile Glu Glu Pro Ile Glu Lys Leu Ser Lys Arg His Glu Arg His305 310 315 320Ile Lys Ala Tyr Tyr Pro Lys Glu Gly Lys Asp Asn Glu Arg Arg Leu 325 330 335Val Gly Arg Leu Glu Thr Ser Ser Ile Asp Lys Phe Ser Trp Gly Val 340 345 350Ala Asn Arg Ala Val Ser Val Arg Val Pro Arg Gly Val Ala Thr Ala 355 360 365Gly Lys Gly Tyr Leu Glu Asp Arg Arg Pro Ser Ser Asn Cys Asp Pro 370 375 380Tyr Ala Val Cys Asn Ala Ile Val Gln Thr Cys Leu Leu Asn Glu385 390 39550403PRTSqualus acanthia 50Met Arg Ile Cys Arg Ser Phe Leu Phe Leu Val Lys Lys Cys Gly Asn1 5 10 15Ile Thr Pro Thr Ile Trp Arg Asn Gln His Thr Tyr Lys Met Ala Thr 20 25 30Ser Ala Ser Ala Asn Leu Ser Lys Ile Val Lys Lys Asn Tyr Met Glu 35 40 45Leu Pro Gln Asp Gly Lys Val Gln Ala Met Tyr Ile Trp Ile Asp Gly 50 55 60Thr Gly Glu Ala Val Arg Cys Lys Thr Arg Thr Leu Asp Asn Glu Pro65 70 75 80Lys Ser Ile Ala Glu Leu Pro Glu Trp Asn Phe Asp Gly Ser Ser Thr 85 90 95Tyr Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Val Pro Ser Ala 100 105 110Met Phe Arg Asp Pro Phe Arg Arg Asp Pro Asn Lys Leu Val Leu Cys 115 120 125Glu Val Leu Lys Tyr Asn Arg Lys Pro Ala Glu Ser Asn Leu Arg His 130 135 140Ser Cys Gln Lys Ile Met Ser Met Ile Ala Asn Glu Tyr Pro Trp Phe145 150 155 160Gly Met Glu Gln Glu Tyr Thr Leu Leu Gly Thr Asp Gly His Pro Phe 165 170 175Gly Trp Pro Ser Asn Cys Phe Pro Gly Pro Gln Gly Pro Tyr Tyr Cys 180 185 190Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val Glu Ala His 195 200 205Tyr Arg Ala Cys Leu Tyr Ala Gly Ile Glu Leu Ser Gly Thr Asn Ala 210 215 220Glu Val Met Ala Ala Gln Trp Glu Tyr Gln Val Gly Pro Cys Glu Gly225 230 235 240Ile Gln Met Gly Asp His Leu Trp Ile Ser Arg Phe Ile Leu His Arg 245 250 255Val Cys Glu Asp Phe Gly Ile Ile Ala Ser Phe Asp Pro Lys Pro Ile 260 265 270Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe Ser Thr Lys 275 280 285Ala Met Arg Asp Asp Gly Gly Leu Lys Tyr Ile Glu Asp Ser Ile Glu 290 295 300Lys Leu Gly Lys Arg His Gln Tyr His Ile Arg Ala Tyr Asp Pro Lys305 310 315 320Gly Gly Leu Asp Asn Ala Arg Ala Leu Thr Gly His His Glu Thr Ser 325 330 335Asn Ile Asn Glu Phe Ser Ala Gly Val Ala Asn Arg Gly Ala Ser Ile 340 345 350Arg Ile Pro Arg Ser Val Gly Gln Asp Lys Lys Gly Tyr Phe Glu Asp 355 360 365Arg Arg Pro Ser Ala Asn Cys Asp Pro Tyr Ala Val Thr Glu Ala Leu 370 375 380Val Arg Thr Cys Leu Leu Asp Glu Ser Gly Asp Lys Pro Ile Glu Tyr385 390 395 400Asn Lys Asn51392PRTXenopus laevis 51Met Ser Val Ser His Ser Ser Arg Leu Asn Lys Gly Val Arg Glu Gln1 5 10 15Tyr Met Lys Leu Pro Gln Gly Glu Lys Val Gln Val Thr Tyr Val Trp 20 25 30Ile Asp Gly Thr Gly Glu Gly Val Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45Gln Glu Pro Lys Thr Ile Asp Glu Ile Pro Glu Trp Asn Phe Asp Gly 50 55 60Ser Ser Thr His Gln Ala Glu Gly Ser Asn Ser Asp Met Tyr Leu Ile65 70 75 80Pro Val Gln Met Phe Arg Asp Pro Phe Cys Leu Asp Pro Asn Lys Leu 85 90 95Val Met Cys Glu Val Leu Lys Tyr Asn Arg Lys Ser Ala Glu Thr Asn 100 105 110Leu Arg His Thr Cys Lys Lys Ile Met Glu Met Val Asn Asp His Arg 115 120 125Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Leu Gly Ile Asn Gly 130 135 140His Pro Tyr Gly Trp Pro Glu Asn Gly Phe Pro Gly Pro Gln Gly Pro145 150 155 160Tyr Tyr Cys Gly Val Gly Ala Asp Lys Val Tyr Gly Arg Asp Val Val 165 170 175Glu Ser His Tyr Lys Ala Cys Leu Tyr Ala Gly Ile Lys Ile Cys Gly 180 185 190Thr Asn Ala Glu Val Met Pro Ser Gln Trp Glu Phe Gln Val Gly Pro 195 200 205Cys Glu Gly Ile Asp Met Gly Asp His Leu Trp Met Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Glu Asp Phe Gly Val Val Ala Thr Leu Asp Pro225 230 235 240Lys Pro Met Thr Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Tyr 245 250 255Ser Thr Glu Ser Met Arg Val Glu Gly Gly Leu Lys His Ile Glu Asp 260 265 270Ala Ile Glu Lys Leu Gly Lys Arg His Asp Tyr His Ile Cys Val Tyr 275 280 285Asp Pro Arg Gly Gly Lys Asp Asn Ser Arg Arg Leu Thr Gly Gln His 290 295 300Glu Thr Ser Ser Ile His Glu Phe Ser Ala Gly Val Ala Asn Arg Gly305 310 315 320Ala Ser Ile Arg Ile Pro Arg Gln Val Gly Gln Glu Gly Tyr Gly Tyr 325 330 335Phe Glu Asp Arg Arg Pro Ala Ala Asn Cys Asp Pro Tyr Ala Val Thr 340 345 350Glu Ala Leu Val Arg Thr Thr Ile Leu Asn Glu Thr Gly Ser Glu Thr 355 360 365Lys Asp Tyr Lys Asn Gly Ala Gly Phe Ser Arg Ala Ile Gly Met Ala 370 375 380Ser Pro Arg Asp Ala Ala Val Phe385 39052373PRTGallus gallus 52Met Ala Thr Ser Ala Ser Ser His Leu Ser Lys Ala Ile Lys His Met1 5 10 15Tyr Met Lys Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30Ile Asp Gly Thr Gly Glu His Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45His Glu Pro Lys Ser Leu Glu Asp Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60Ser Ser Thr Phe Gln Ala Glu Gly Ser Asn Ser Asp Met Tyr Leu Arg65 70 75 80Pro Ala Ala Met Phe Arg Asp Pro Phe Arg Lys Asp Pro Asn Lys Leu 85 90 95Val Leu Cys Glu Val Phe Lys Tyr Asn Arg Gln Ser Ala Asp Thr Asn 100 105 110Leu Arg His Thr Cys Arg Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Leu Gly Thr Asp Gly 130 135 140His Pro Phe Gly Trp Pro Ser Asn Cys Phe Pro Gly Pro Gln Gly Pro145 150 155 160Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val 165 170 175Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Gly Gly 180 185 190Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Val Gly Pro 195 200 205Cys Glu Gly Ile Glu Met Gly Asp His Leu Trp Ile Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Val Ser Phe Asp Pro225 230 235 240Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245 250 255Ser Thr Lys Asn Met Arg Glu Asp Gly Gly Leu Lys His Ile Glu Glu 260 265 270Ala Ile Glu Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280 285Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His 290 295 300Glu Thr Ser Ser Ile His Glu Phe Ser Ala Gly Val Ala Asn Arg Gly305 310 315 320Ala Ser Ile Arg Ile Pro Arg Asn Val Gly His Glu Lys Lys Gly Tyr 325 330 335Phe Glu Asp Arg Gly Pro Ser Ala Asn Cys Asp Pro Tyr Ala Val Thr 340 345 350Glu Ala Leu Val Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365Phe Glu Tyr Lys Asn 37053373PRTMus musculus 53Met Ala Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Met1 5 10 15Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30Val Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45Cys Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60Ser Ser Thr Phe Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu His65 70 75 80Pro Val Ala Met Phe Arg Asp Pro Phe Arg Arg Asp Pro Asn Lys Leu 85 90 95Val Leu Cys Glu Val Phe Lys Tyr Asn Arg Lys Pro Ala Glu Thr Asn 100 105 110Leu Arg His Ile Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro145 150 155 160Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala Tyr Gly Arg Asp Ile Val 165 170 175Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Thr Gly 180 185 190Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro 195 200 205Cys Glu Gly Ile Arg Met Gly Asp His Leu Trp Ile Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Ala Thr Phe Asp Pro225 230 235 240Lys Pro Ile Pro Gly Asn Trp Asn Val Ala Gly Cys His Thr Asn Phe 245 250 255Ser Thr Lys Ala Met Arg Glu Glu Asn Gly Leu Lys Cys Ile Glu Glu 260 265 270Ala Ile Asp Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280 285Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Ala Leu Thr Gly Phe His 290 295 300Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Gly305 310 315 320Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr 325 330 335Phe Glu Asp Arg Arg Leu Arg Ala Asn Cys Asp Pro Tyr Ala Val Thr 340 345 350Glu Ala Ile Val Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365Phe Gln Tyr Lys Asn 37054373PRTCricetulus griseus 54Met Ala Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Met1 5 10 15Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30Val Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45Cys Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60Ser Ser Thr Phe Gln Ser Glu Ser Ser Asn Ser Asp Met Tyr Leu Ser65 70 75 80Pro Val Ala Met Phe Arg Asp Pro Phe Arg Lys Glu Pro Asn Lys Leu 85 90 95Val Phe Cys Glu Val Phe Lys Tyr Asn Gln Lys Pro Ala Glu Thr Asn 100 105 110Leu Arg His Thr Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Leu Gly Thr Asp Gly 130 135 140His Pro Phe Gly Trp Pro Ser Asp Gly Phe Pro Gly Pro Gln Gly Leu145 150 155 160Tyr Tyr Cys Gly Val Gly Ala Asp Lys Ala Tyr Arg Arg Asp Ile Met 165 170 175Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Thr Gly 180 185 190Thr Tyr Ala Glu Val Lys His Ala Gln Trp Glu Phe Gln Ile Gly Pro 195 200 205Cys Glu Gly Ile Arg Met Gly Asp His Leu Trp Val Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Lys Asp Phe Gly Val Ile Ala Thr Phe Asp Ser225 230 235 240Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245 250 255Ser Thr Lys Thr Met Arg Glu Glu Asn Gly Leu Lys His Ile Lys Glu 260 265 270Ala Ile Glu Lys Leu Ser Lys Arg His Arg Tyr His Ile Arg Ala Tyr 275 280 285Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His 290 295 300Lys Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asp Arg Ser305 310 315 320Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr 325 330 335Phe Glu Ala Arg Cys Pro Ser Ala Asn Cys Asp Pro Phe Ala Val Thr 340 345 350Glu Ala Ile Val Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Gln Pro 355 360 365Phe Gln Tyr Lys Asn 37055373PRTHomo sapiens 55Met Thr Thr Ser Ala Ser Ser His Leu Asn Lys Gly Ile Lys Gln Val1 5 10 15Tyr Met Ser Leu Pro Gln Gly Glu Lys Val Gln Ala Met Tyr Ile Trp 20 25 30Ile Asp Gly Thr Gly Glu Gly Leu Arg Cys Lys Thr Arg Thr Leu Asp 35 40 45Ser Glu Pro Lys Cys Val Glu Glu Leu Pro Glu Trp Asn Phe Asp Gly 50 55 60Ser Ser Thr Leu Gln Ser Glu Gly Ser Asn Ser Asp Met Tyr Leu Val65 70 75 80Pro Ala Ala Met Phe Arg Asp Pro Phe Arg Lys Asp Pro Asn Lys Leu 85 90 95Val Leu Cys Glu Val Phe Lys Tyr Asn Arg Arg Pro Ala Glu Thr Asn 100 105 110Leu Arg His Thr Cys Lys Arg Ile Met Asp Met Val Ser Asn Gln His 115 120 125Pro Trp Phe Gly Met Glu Gln Glu Tyr Thr Leu Met Gly Thr Asp Gly 130 135 140His Pro Phe Gly Trp Pro Ser Asn Gly Phe Pro Gly Pro Gln Gly Pro145 150 155 160Tyr Tyr Cys Gly Val Gly Ala Asp Arg Ala Tyr Gly Arg Asp Ile Val 165 170 175Glu Ala His Tyr Arg Ala Cys Leu Tyr Ala Gly Val Lys Ile Ala Gly 180 185 190Thr Asn Ala Glu Val Met Pro Ala Gln Trp Glu Phe Gln Ile Gly Pro 195 200 205Cys Glu Gly Ile Ser Met Gly Asp His Leu Trp Val Ala Arg Phe Ile 210 215 220Leu His Arg Val Cys Glu Asp Phe Gly Val Ile Ala Thr Phe Asp Pro225 230 235 240Lys Pro Ile Pro Gly Asn Trp Asn Gly Ala Gly Cys His Thr Asn Phe 245

250 255Ser Thr Lys Ala Met Arg Glu Glu Asn Gly Leu Lys Tyr Ile Glu Glu 260 265 270Ala Ile Glu Lys Leu Ser Lys Arg His Gln Tyr His Ile Arg Ala Tyr 275 280 285Asp Pro Lys Gly Gly Leu Asp Asn Ala Arg Arg Leu Thr Gly Phe His 290 295 300Glu Thr Ser Asn Ile Asn Asp Phe Ser Ala Gly Val Ala Asn Arg Ser305 310 315 320Ala Ser Ile Arg Ile Pro Arg Thr Val Gly Gln Glu Lys Lys Gly Tyr 325 330 335Phe Glu Asp Arg Arg Pro Ser Ala Asn Cys Asp Pro Phe Ser Val Thr 340 345 350Glu Ala Leu Ile Arg Thr Cys Leu Leu Asn Glu Thr Gly Asp Glu Pro 355 360 365Phe Gln Tyr Lys Asn 3705680DNASynthetic oligo 56gaggatccca gaaccagcgc catcagcgtt accatggcac cagctacaac cttgaaccaa 60ttaaccctca ctaaagggcg 805781DNASynthetic oligo 57gtggatccgc gatatcgtga aacagcgcgg cgatgaaaat cagctcagtt gacggcagta 60atacgactca ctatagggct c 815880DNASynthetic oligo 58gaggatcctg cgcctgtttg aactgacgca gcgcctcaag ctgttgctct tcgtcatcaa 60ttaaccctca ctaaagggcg 805981DNASynthetic oligo 59gtggatccag gtgttccagc tcattcgcgg cggacgcgaa ccgtcgctgc aatcgcgcta 60atacgactca ctatagggct c 81

Claims

1. A method of inhibiting the activity of a kinase or a synthetase, the method including binding an active site of the kinase or synthetase with a deuterated imidazole moiety, thereby inhibiting the activity of the kinase or the synthetase.

2. The method of claim 1, wherein it is applied to a kinase or synthetase, and wherein the kinase is selected from the group consisting of adenylate kinase (AK), shikimate kinase (SK), pyruvate kinase (PK), hexokinase (HXK), aspartokinase (ASK), creatine kinase (CK), glycerate kinase, acetate kinase, phosphofructokinase and the synthetase is glutamine synthetase, such as adenylated glutamine synthetase, and/or deadenylated glutamine synthetase, and isoforms thereof.

3. The method of claim 2, wherein the kinase is adenylate kinase, whose sequence comprises conserved residues Arg 97, Glu 98, Arg 128 and Asp 180.

4. The method of claim 1, wherein the kinase or the synthetase, or an isoenzyme thereof, comprises amino acid residues identical to, or similar to, conserved adenylate kinase residues Arg 97, Glu 98, Arg 128 and Asp 180, at positions equivalent to the positions of these residues in adenylate kinase.

5. The method of any one of claim 1, wherein the inhibition of the kinase or the synthetase activity is effected in vitro.

6. The method of claim 1, wherein the deuterated imidazole moiety is provided by a nucleotide.

7. The method of claim 6, wherein the nucleotide is adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP), with the nucleotide having the C8-H induced to be more acidic, and with deuteration being effected at the C8 position.

8. The method of claim 6, wherein the nucleotide is a compound which is deuterated at a position equivalent to C8 in ATP, ADP and AMP.

9. A method of coupling a kinase or a synthetase to a nucleotide or to a nucleotide analogue, to inhibit the activity of the kinase or the synthetase, the method including binding an active site of the kinase or the synthetase with a nucleotide or with a nucleotide analogue comprising an imidazole moiety in deuterated form.

10. A method of generating a compound that inhibits the activity of a kinase or a synthetase, the method comprising providing a three-dimensional structure of a kinase or a synthetase; and designing, based on the three-dimensional structure, a compound capable of inhibiting the activity of the kinase or the synthetase, the compound comprising a deuterated imidazole moiety.

11. A computer-assisted method of generating a test inhibitor of the activity of a kinase or a synthetase, the method using a processor and an input device, the method comprising (a) inputting, on the input device, data comprising a structure of a kinase or a synthetase; (b) docking into an active site of the kinase or the synthetase, a test inhibitor molecule comprising a deuterated imidazole moiety, using the processor; and (c) determining, based on the docking, whether the test inhibitor compound would inhibit the kinase or synthetase activity.

12. The method of claim 11, which includes determining, based on the docking, whether the test inhibitor molecule would inhibit the transfer of a γ-phosphate group from a phosphate donor.

13. The method of claim 11, which further comprises designing a test inhibitor determined by step (c) to inhibit the kinase or the synthetase activity and evaluating the inhibitory activity of the test inhibitor on a bacterial kinase or synthetase in vitro.

14. A method of screening a compound in vitro to determine whether or not it inhibits the activity of kinase or a synthetase, the method comprising contacting a kinase or a synthetase with a compound comprising a protonated imidazole moiety; contacting the kinase or the synthetase with the same compound comprising a deuterated imidazole moiety; and determining whether or not the activity of the kinase or synthetase is reduced in the presence of the compound containing the deuterated imidazole moiety relative to the activity of the same kinase or synthetase in the presence of the compound containing the protonated imidazole moiety.

15. The method of claim 9 wherein it is applied to a kinase, and, wherein the kinase is selected from a group consisting of adenylate kinase (AK), shikimate kinase (SK), pyruvate kinase (PK), hexokinase (HXK), aspartokinase (ASK), creatine kinase (CK), glycerate kinase, acetate kinase, phosphofructokinase, and the synthetase is glutamine synthetase, such as adenylated glutamine synthetase, and/or deadenylated glutamine synthetase, and isoforms thereof.

16. The method of claim 9, wherein the deuterated imidazole moiety is provided by a nucleotide or by a nucleotide analogue.

17. The method of claim 16, wherein the nucleotide is adenosine triphosphate (ATP), adenosine diphosphate (ADP) or adenosine monophosphate (AMP), with the nucleotide having an immonium moiety at position N7, so that a carbene is induced at the C8 position, and with deuteration being effected at the C8 position.

18. The method of claim 16, wherein the nucleotide is a compound which is deuterated at a position equivalent to C8 in ATP, ADP and AMP.

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